Method of promoting mucosal reciliation

ABSTRACT

The present invention relates to ionic compositions for promoting mucosal reciliation, particularly in the sinonasal cavity. The ionic components of the composition can include potassium, calcium, rubidium, zinc, bromide, and magnesium in an isotonic solution. In other embodiments, the ionic components of the composition can include magnesium, bromide, sulfate, sodium, and chloride.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of and claims priority to currentlypending U.S. Nonprovisional application Ser. No. 14/162,112, entitled“Methods of Enhancing Wound Healing”, filed Jan. 23, 2014, which is adivisional of and claims priority to U.S. Nonprovisional applicationSer. No. 13/460,426, entitled “Compositions and Methods of EnhancingWound Healing”, filed Apr. 30, 2012, which claims priority to U.S.Provisional Application No. 61/480,764 entitled “Compositions andMethods of Enhancing Wound Healing”, filed Apr. 29, 2011, the contentsof each of which are hereby incorporated by reference into thisdisclosure.

FIELD OF INVENTION

This invention relates to promoting mucosal reciliation. Specifically,the invention provides compositions and methods to promote mucosalreciliation in epithelial mucosal tissue such as in the sinonasalregion.

BACKGROUND OF INVENTION Nasal Cavity

The nasal cavity is the body's first line of defense of the respiratorysystem that filters and removes airborne vapors, particles, pollutants,and toxins that are inhaled. The nasal passages are normally aself-cleaning structure that purifies and humidifies inhaled air priorto delivery of the vital gases to the lungs. Particles having about 5 umaerodynamic equivalent diameter (AED) or greater are normally removed bythe nose and nasopharynx (about 85-90% are removed). Smaller particlesmay penetrate the lower respiratory tract to varying degrees.

The nasal passages are lined with a semi-permeable mucous membraneepithelium. The glands in each passage secrete protective fluids thatkeep the microbial flora in balance and protect against colonization andinfection by overgrowth of surface microbes. The natural liquid productsmaintain a specific pH level to support the metabolic requirement oflocal host tissues while being unfavorable to the survival of microbes.In healthy individuals, the normal intranasal pH level of the mucousranges from about 4 to about 7. The mucous generally is comprised ofmucin of which about 2.5-3% is glycoprotein; about 1-2% is salts; andabout 95% is water. Immunoglobulins comprise about 70% of the proteincontent.

Chronic Rhinosinusitis

Chronic rhinosinusitis is a common disorder that affects approximately13% of the population in the United States. Chronic rhinosinusitis is aninflammatory disease of the mucosal lining of the sinuses and nasalcavity. Chronic rhinosinusitis symptoms include nasal congestion, facialpain, headache, nighttime coughing, an increase in previously minor orcontrolled asthma symptoms, general malaise, thick green or yellowdischarge, feeling of facial fullness or tightness that may worsen whenbending over, dizziness and aching teeth. A common treatment for chronicrhinosinusitis is functional endoscopic sinus surgery. In some cases,antibiotic treatment is also prescribed.

Wound Healing of the Nasal Mucosa

The sinonasal cavity is lined by pseudostratified columnar ciliatedepithelium. The epithelium has a variable number of ciliated cells(˜75%), goblet cells (˜20%), and basal cells (˜5%), which reside on anacellular basement membrane. This epithelial lining protects the upperairway from inhaled pathogens and debris by a process referred to asmucociliary clearance and contributes to the innate immunity and antigenpresentation defense mechanisms. (Schleimer R P, Kato A, Kern R, et al.Epithelium: at the interface of innate and adaptive immune responses. JAllergy Clin Immunol. 2007; 120:1279-1284). Performance is dependent ona complex interaction between motile cilia, glandular secretions andsinus anatomy. (Huang H M, Cheng J J, Liu C M, et al. Mucosal healingand mucociliary transport change after endoscopic sinus surgery inchildren with chronic maxillary sinusitis. Int J PediatrOtorhinolaryngol. 2006; 70:1361-1367).

Chronic rhinosinusitis is a common disorder that affects approximately13% of the population in the United States. Chronic rhinosinusitisresults from inflammation of the sinonasal mucosa. Its symptoms include:nasal congestion; facial pain; headache; nighttime coughing; an increasein previously minor or controlled asthma symptoms; general malaise;thick green or yellow discharge; a feeling of facial fullness ortightness that may worsen when bending over; dizziness; and achingteeth. It should be stressed that the underlying etiology of theinflammation is multifactoral, with varying contributions fromcomponents of genetic predisposition as well as environmental exposures.(Kennedy D W. Pathogenesis of chronic rhinosinusitis. Ann Otol RhinolLaryngol Suppl. 2004; 193:6-9.) However, invariable of the etiologicsource, the common pathophysiologic endpoint is impaired mucociliaryclearance with stagnant sinonasal secretions. (Gudis D A, Cohen N A.Cilia dysfunction. Otolaryngol Clin North Am. 2010; 43:461-472, vii).

Management of the disease is primarily focused on restoring mucociliaryfunction and is typically accomplished with antimicrobial therapy(bacterial/fungal), anti-inflammatory therapy (topical or systemicsteroids), and sinonasal lavage. While this combination approach iseffective in a vast array of patients a subset of individuals requiresurgical intervention to relieve obstruction and restore mucociliaryclearance.

Functional endoscopic sinus surgery (FESS) is a minimally invasivetechnique developed to restore the natural mucus clearance pathways.(Kennedy D W, Zinreich S J, Rosenbaum A E, et al. Functional endoscopicsinus surgery. Theory and diagnostic evaluation. Arch Otolaryngol. 1985;111:576-582). As this technique has gained popularity, experience hasdemonstrated the importance of mucosal preservation; i.e., areas denudedof mucosa with exposed bone tend to develop osteitis with persistentinflammation. (Moriyama H, Yanagi K, Ohtori N, et al. Healing process ofsinus mucosa after endoscopic sinus surgery. Am J Rhinol. 1996;10:61-66). Additionally, these areas tend to demonstrate abnormalmucosal regeneration with inadequate mucociliary clearance. (Shaw C K,Cowin A, Wormald P J. A study of the normal temporal healing pattern andthe mucociliary transport after endoscopic partial and full-thicknessremoval of nasal mucosa in sheep. Immunol Cell Biol. 2001; 79:145-148).

Uncertainty still exists concerning the sequence and time required forrespiratory mucosa to heal. Differences in the extent and depth oftrauma, animal species, and the criteria used to evaluate data mayexplain the disparate results reported. (Forsgren K, Stierna P, KumlienJ, et al. Regeneration of maxillary sinus mucosa following surgicalremoval. Experimental study in rabbits. Ann Otol Rhinol Laryngol. 1993;102:459-466).

Wound healing is a highly organized process. Fibroblasts, leukocytes,and epithelial cells regulated by a wide variety of growth factors andcytokines interact resulting in inflammation, extracellular matrix (ECM)deposition and remodeling, cell migration, replication, anddifferentiation. A disturbance in this sequence can affect the normalfunction of the organ involved. (Watelet J B, Bachert C, Gevaert P, etal. Wound healing of the nasal and paranasal mucosa: a review. Am JRhinol. 2002; 16:77-84). The process of wound repair has beenextensively studied in other tissues such as the gingiva and the dermis,but epithelial wound repair in the nose and paranasal sinuses is stillpoorly understood. (Zahm J M, Chevillard M, Puchelle E. Wound repair ofhuman surface respiratory epithelium. Am J Respir Cell Mol Biol. 1991;5:242-248).

Injury to the nasal epithelium causes hemorrhage and exposes connectivetissue. Subsequent vasoconstriction and platelet aggregation helpcontrol bleeding while fibrin and fibronectin, act as a provisionalmatrix for the influx of fibroblasts and monocytes. An intenseinflammatory reaction is initiated simultaneously with the coagulationphase with neutrophils initially predominating. Three days after theinjury the neutrophilic population is replaced by macrophages. (WateletJ B, Bachert C, Gevaert P, et al. Wound healing of the nasal andparanasal mucosa: a review. Am J Rhinol. 2002; 16:77-84).

Cell migration after wounding is an important process by which therespiratory epithelial barrier integrity is maintained. (Zahm J M,Pierrot D, Chevillard M, et al. Dynamics of cell movement during thewound repair of human surface respiratory epithelium. Biorheology. 1992;29:459-465). The movement of respiratory cells from around the woundedge initiates within a few hours and is uniformly distributed. Toinitiate migration the apical-basal polarity of the cell is lost andcytoplasmatic extensions known as lamellipodia develop. (Watelet J B,Bachert C, Gevaert P, et al. Wound healing of the nasal and paranasalmucosa: a review. Am J Rhinol. 2002; 16:77-84). Small-size injuries mayheal with no need for mitosis to occur, demonstrated by the use ofcolchicine, a mitosis inhibitor, which did not interrupt the repairprocess and cytochalasin B, an actin inhibitor, which prevented cellmigration. (Zahm J M, Chevillard M, Puchelle E. Wound repair of humansurface respiratory epithelium. Am J Respir Cell Mol Biol. 1991;5:242-248). When replication is needed, undifferentiated basal cellsfrom adjacent untraumatized areas are the main source of nascent cells.(Forsgren K, Stierna P, Kumlien J, et al. Regeneration of maxillarysinus mucosa following surgical removal. Experimental study in rabbits.Ann Otol Rhinol Laryngol. 1993; 102:459-466). Reorientation anddifferentiation follow while it appears that cilia require a reasonablebase of epithelium before reciliation occurs and thus represent terminaldifferentiation. (Shaw C K, Cowin A, Wormald P J. A study of the normaltemporal healing pattern and the mucociliary transport after endoscopicpartial and full-thickness removal of nasal mucosa in sheep. ImmunolCell Biol. 2001; 79:145-148).

Postoperative outcomes from FESS are directly dependent on the healingquality of the nasal mucosa. (Watelet J B, Bachert C, Gevaert P, et al.Wound healing of the nasal and paranasal mucosa: a review. Am J Rhinol.2002; 16:77-84). The ECM has a profound influence on cell adhesion andmigration. Epithelial respiratory cells are able to retract collagen,transmitting contractile forces to the collagen fibrils. (Zahm J M,Pierrot D, Chevillard M, et al. Dynamics of cell movement during thewound repair of human surface respiratory epithelium. Biorheology. 1992;29:459-465; Benali R, Tournier J M, Chevillard M, et al. Tubuleformation by human surface respiratory epithelial cells cultured in athree-dimensional collagen lattice. Am J Physiol. 1993; 264:L183-L192).

Matrix Metalloproteinases (MMPs)

Matrix metalloproteinases (MMPs) are a family of secreted proteolyticenzymes that use ECM as a substrate and remodel the ECM which is acritical step in wound healing. It is now apparent that MMPs play animportant role for many of the normal or pathological processesrequiring matrix turnover. (Buisson A C, Zahm J M, Polette M, et al.Gelatinase B is involved in the in vitro wound repair of humanrespiratory epithelium. J Cell Physiol. 1996; 166:413-426).

Metalloproteinase-9 (MMP-9) expression is increased in the ECM and nasalfluids during wound healing, and is predominately derived frominflammatory cells, mainly neutrophils and macrophages. MMP-9 is knownto actively degrade collagen, fibronectin, and elastin, subsequentlyinterfering with the repair process. MMP-9 activity is controlled atmultiple levels: transcriptional control by various cytokines,activation of the proenzyme by serine proteases or othermetalloproteinases, and activity regulation by natural tissue inhibitorsof metalloproteinases (TIMPs). It was reported that post-functionalsinus surgery (post-FESS) patients with poor healing, characterized byedema, inflammation, and fibrosis, presented higher expression of MMP-9in the ECM than those with good healing. (Watelet J B, Demetter P,Claeys C, et al. Neutrophil derived metalloproteinase-9 predicts healingquality after sinus surgery. Laryngoscope. 2005; 115:56-61).

Ions in Wound Healing

Various metal ions have been shown to be therapeutic in many diseases ofman. For example, transition metal ions such as copper, iron, manganeseand zinc serve as essential cofactors for a variety of biologicalprocesses including: cell energetics, gene regulation, and control offree radicals.

Potassium

Potassium, an electrolyte, is important for the proper function of allcells, tissues and organs in the human body. Potassium is necessary forproper heart function and plays a key role in skeletal and smooth musclecontraction, thus making it important for normal digestive and muscularfunction. Moderate to severe hypokalemia, or low plasma potassiumlevels, must often be redressed with oral potassium chloridesupplementation or intravenous supplementation.

Magnesium

Magnesium is another ion that is necessary for more than 300 biochemicalreactions in the body. Magnesium assists in maintaining normal muscleand nerve function; maintaining a steady heart rhythm; supporting ahealthy immune system; maintaining strong bones; maintaining the normalfunctioning of enzymes; regulating blood sugar levels; regulating bloodpressure; synthesizing proteins; maintaining the normal functioning ofmany hormones, particularly parathyroid hormone; and modulating energymetabolism. There is an increased interest in the role of magnesium inpreventing and managing disorders such as hypertension, cardiovasculardisease, and diabetes.

Changes in plasma magnesium levels usually affect the levels of othermetal ions. For example, magnesium deficiency (hypomagnesemia) can causelow calcium and/or potassium levels. Symptoms of hypomagnesemia includemuscle cramps and weakness, tremors, and abnormal heart rhythms. If themagnesium level is only a little low, the condition can be treated withoral magnesium tablets or by intramuscular injection. If the magnesiumlevel is very low a large dose of magnesium can be given intravenouslyover a short time. Magnesium sulfate injections are also used to preventpremature contractions and to control seizures in pregnancy, to treatproblems related to kidney conditions in children, and to treat heartattack and asthma patients.

Calcium

Calcium is another metal ion that is essential to maintaining total bodyhealth. Calcium is necessary for maintaining bones and teeth andensuring proper functioning of muscles and nerves. Calcium deficiencycan make the nervous system highly irritable causing tetany (spasms ofthe hands and feet, muscle cramps, abdominal cramps, and overly activereflexes). Chronic calcium deficiency contributes to poor mineralizationof bones, soft bones (osteomalacia) and osteoporosis and, in children,rickets and impaired growth.

Calcium also plays a pivotal role in the physiology and biochemistry ofthe cell. For example, calcium acts as a second messenger in signaltransduction pathways, in neurotransmitter release from neurons,contraction of all muscle cell types, and fertilization. Many enzymesrequire calcium ions as a cofactor, especially those of theblood-clotting cascade. Extracellular calcium is also important formaintaining the potential difference across excitable cell membranes.

Rubidium

Rubidium is a metal ion that is relatively safe with no known toxiceffects. Rubidium has been used in therapy for chronic mental conditionsin humans such as depression. It has a mild tranquilizing effect, andcan be administered to those with nervous disorders and epilepsy.Rubidium is chemically similar to potassium and cesium, and is one ofthe few compounds available that is capable of entering cancer cells.Due to this ability and its pH, it is used in high pH cancer therapies.Some studies have shown that rubidium therapy can shrink tumors, byreplacing the potassium that allows the cells to replicate. Rubidium hasalso been shown to regulate cell membranes; regulate insulin levels; aidin digestion of proteins; aid in the release of hormones from thepituitary gland; control release of fluids from the salivary glands; andaid in regulating heartbeat. A lack of rubidium can cause depression,dehydration of the cells, susceptibility to cancer, hair loss, anddecreased tolerance to glucose.

Zinc

Zinc is one of the essential trace elements in humans and is ubiquitousin subcellular metabolism. It is an essential component of at least onecatalytic site of at least one enzyme in every enzyme classification.Altogether, several hundred zinc metalloenzymes have been identified inthe plant and animal kingdoms, including matrix metalloproteins that areimportant in wound healing.

Wound healing solutions containing cations, such as polyhydrated ionogensolution (PHI), were originally based on constituents found in red oaktree bark which was used as a traditional wound dressing of the NativeAmerican Indians. The original cation containing solutions were based ona combination of citric acid and metallic ions such as potassium,calcium, rubidium and zinc. The formulation is aimed at modulatingextracellular protease activity, altering reactive oxygen species andmodulating expression of inflammatory cytokines (Monroe S, Sampson E M,Popp M P. Effect of Polyhydrated Ionogen (PHI) on Viability and MatrixMetalloproteinase Levels in Cultures of Normal and Diabetic Human DermalFibroblasts. Chicago, Ill.: Wound Healing Society; 2005; van den Berg AJ J, Halkes S B A, Quarles van Ufford H C, et al. A novel formulation ofmetal ions and citric acid reduces reactive oxygen species in vitro.Wound Care. 2003; 12:413-418). The formulation has been demonstrated toimprove healing in chronic wounds such as diabetic foot ulcers.(Pirayesh A, Dessy L A, Rogge F J, et al. The efficacy of a polyhydratedionogen impregnated dressing in the treatment of recalcitrant diabeticfoot ulcers: a multicentre pilot study. Acta Chir Belg. 2007;107:675-681).

The use of MgBr2 for dermatologic issues such as psoriasis dates back tothe ancient Egyptians and has demonstrated inhibitory effects onfibroblast outgrowth. (Proksch E, Nissen H P, Bremgartner M, et al.Bathing in a magnesium-rich Dead Sea salt solution improves skin barrierfunction, enhances skin hydration, and reduces inflammation in atopicdry skin. Int J Dermatol. 2005; 44:151-157; Levi-Schaffer F, Shani J,Politi Y, et al Inhibition of proliferation of psoriatic and healthyfibroblasts in cell culture by selected Dead-sea salts. Pharmacology.1996; 52:321-328).

Given the difficulty in successfully healing wounds in the mucosa of thenasal cavity, what is needed is a composition that enhances woundhealing and promotes recilialization in the mucosa.

SUMMARY OF THE INVENTION

The present application determined that the therapeutic potential of acation containing formulation realized in dermatologic wounds could betranslated to sinonasal mucosal healing. A novel combination of metalions that is safe and exhibits unanticipated additive/synergisticeffects in the efficacy of treatment of inflamed and injured mucosaltissue and sinuses is presented. The combination of ions such aspotassium (K+), rubidium (Rb+), magnesium, (Mg2+), calcium (Ca2+), zinc(Zn2+), and bromide (Br−) with a pharmaceutically acceptable carrier inan isotonic solution provides for enhanced healing of damaged mucosaltissue over the treatment of this form of injury by any one of the metalions alone and relative to the current standard of care by normal salinesolution (buffered or unbuffered). An effective amount of citrate and/orcitric acid as well as sulfate may also be added to the solution of thepresent invention. Water may be the pharmaceutically acceptable carrier.This solution may be administered at least once per day in an amountfrom between about 0.1 ounces to about 30 ounces per day.

A method of enhanced wound healing is presented in which an isotonicsolution containing an effective amount of K⁺, Rb⁺, Ca²⁺, Zn²⁺, Mg²⁺,SO₄ ²⁻ and Br⁻ ions is administered to a subject at least once per day.Citric acid or citrate may also be added to the composition. Thisenhanced wound healing may be sinonasal mucosal healing and the solutionmay be administered via a nasal spray. The amount administered can befrom between about 0.1 ounces to about 30 ounces per day.

A method of promoting mucosal reciliation is also presented in which anisotonic solution containing an effective amount of K⁺, Rb⁺, Ca²⁺, Zn²⁺,Mg²⁺, SO₄ ²⁻ and Br⁻ ions is administered to a subject at least once perday. Citric acid or citrate may also be added to the composition. In analternative embodiment, a method of promoting mucosal reciliation ispresented in which an isotonic solution containing an effective amountof magnesium (Mg²⁺), bromide (Br⁻), sodium (Na⁺), chloride (Cl⁻), andsulfate (SO₄ ²⁻) without the preservative potassium sorbate isadministered to a subject at least once per day. The amount administeredcan be from between about 0.1 oz. to about 30 oz.

A method of debriding tissue is presented in which an isotonic solutioncontaining an effective amount of K⁺, Rb⁺, Ca²⁺, Zn²⁺, Mg²⁺, SO₄ ²⁻ andBr⁻ ions is administered to a subject at least once per day. Citric acidor citrate may also be added to the composition. The amount administeredcan be from between about 0.1 ounces to about 30 ounces per day.

In addition, an isotonic solution containing magnesium (Mg²⁺), bromide(Br⁻), sodium (Na⁺), chloride (Cl⁻), and sulfate (SO₄ ²⁻) without thepreservative potassium sorbate is also capable of promoting enhancedhealing of injured mucosal tissue when administered at least once perday in an amount from between about 0.1 ounces to about 30 ounces perday.

In an alternative embodiment, a method of enhancing wound healing ispresented in which an isotonic solution containing an effective mount ofmagnesium (Mg²⁺), bromide (Br⁻), sodium (Na⁺), chloride (Cl⁻), andsulfate (SO₄ ²⁻) without the preservative potassium sorbate isadministered to a subject at least once per day. The amount administeredcan be from between about 0.1 ounces to about 30 ounces per day. Thisenhanced wound healing may be sinonasal mucosal healing and the solutionmay be administered via a nasal spray.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a series of images depicting the effect of three various metalion formulations on mouse cilial function. (A) isotonic formulation ofK⁺, Rb⁺, Ca²⁺ and Zn²⁺ (Solution 2) with and without the preservativepotassium sorbate; (B) isotonic formulation of K⁺, Rb⁺, Ca²⁺, Zn²⁺, Mg²⁺and Br⁻ (Solution 3) with and without the preservative potassiumsorbate; (C) hypertonic formulation of K⁺, Rb⁺, Ca²⁺, Zn²⁺, Mg²⁺ and Br⁻(Solution 1 plus Mg²⁺ and Br⁻) with and without the preservativepotassium sorbate.

FIG. 2 is an image depicting the nasal cavity (NC) epithelium ascontrasted against the Left Maxillary Sinus (LMS) after a sinus mucosalstripping procedure in which no treatment is administered. Two dayspost-operatively no epithelia are observed and a fibrous material coversthe sinus walls. Denuded bone (B) is present.

FIG. 3 is an image depicting the nasal cavity seven dayspost-operatively with no treatment administered. A “transitional”squamous epithelium is observed over an inflammatory collagenous matrix.

FIG. 4 is an image of the LMS fifteen days after mucosal removal. Asdepicted in the image, the LMS has been almost obliterated by fibrosisand concentric neo-ontogenesis, with only a small remnant of a lumen.Note the mature bone (B), concentric bone formation (CBF), fibrosis (F)and remnant of lumen (L).

FIG. 5 is an image of the LMS 22 days after mucosal removal illustratingcomplete obliteration of the anterior portion of the LMS.

FIG. 6 is a series of images depicting the nasal cavity two weeks aftermucosal stripping and treatment with either saline or an isotonicsolution containing K⁺, Rb⁺, Ca²⁺, and Zn²⁺ (Solution 2) without thepreservative potassium sorbate. (A) medial wall injury with nointervention; (B) complete mucosal stripping with no intervention; (C)complete mucosal stripping with daily saline irrigation (3 cc); (D)complete mucosal stripping with daily irrigation (3 cc) with a metal ionSolution 2 (sterile isotonic formulation of K⁺, Rb⁺, Ca²⁺, Zn²⁺ withoutthe preservative potassium sorbate).

FIG. 7 is an image of a representative rabbit snout with positions onthe “injured” medial wall indicated (boxes) where four images were takenfor analysis of ciliated remucociliazation. Images were also taken fromthe uninjured lateral wall to represent normal ciliation of the mucosalwall.

FIG. 8 is a series of images from the uninjured lateral wall that areType IV β-tubulin-stained. (A) a maxillary sinus treated with saline and(B) a maxillary sinus treated with a sterile isotonic formulation of K⁺,Rb⁺, Ca²⁺, Zn²⁺ (Solution 2) without the preservative potassium sorbate.

FIG. 9 is a series of images from the injured upper medial wall that areType IV β-tubulin-stained. (A) a maxillary sinus treated with saline and(B) a maxillary sinus treated with a sterile isotonic formulation of K⁺,Rb⁺, Ca²⁺, Zn²⁺ (solution 2) without the preservative potassium sorbate.As shown in (B), daily treatment with the isotonic formulation of K⁺,Rb⁺, Ca²⁺, Zn²⁺ (Solution 2) without the preservative potassium sorbatehas resulted in recilialization.

FIG. 10 is a series of images from the injured lower medial wall thatare Type IV β-tubulin-stained. (A) a maxillary sinus treated with salineand (B) a maxillary sinus treated with a sterile isotonic formulation ofK⁺, Rb⁺, Ca²⁺, Zn²⁺ (Solution 2) without the preservative potassiumsorbate. As shown in (B), daily treatment with the isotonic formulationof K⁺, Rb⁺, Ca²⁺, Zn²⁺ (Solution 2) without the preservative potassiumsorbate has resulted in recilialization.

FIG. 11 is a series of images from the injured medial wall of the rabbitmaxillary sinus in which sections of the medial maxillary sinus wallwere surgically stripped of mucosal cells and treated for 14 days witheither saline or an isotonic formulation of K⁺, Rb⁺, Ca²⁺, Zn²⁺, Mg²⁺and Br⁻ (Solution 3) without the preservative potassium sorbate. (A) H&Estaining of rabbit maxillary sinus treated with saline; (B) H&E stainingof rabbit maxillary sinus treated with isotonic formulation of K⁺, Rb⁺,Ca²⁺, Zn²⁺, Mg²⁺ and Br⁻ (Solution 3) without the preservative potassiumsorbate; (C) Type IV β-tubulin-staining of rabbit maxillary sinustreated with saline; (D) Type IV β-tubulin-staining of rabbit maxillarysinus treated with isotonic formulation of K⁺, Rb⁺, Ca²⁺, Zn²⁺, Mg²⁺ andBr⁻ (Solution 3) without the preservative potassium sorbate.

FIG. 12 is a series of images depicting a semi quantitative grading ofreciliation. (A) sections of rabbit snout fixed and stained withhematoxylin and eosin; (B) sections of rabbit snout fixed and subjectedto immunohistochemistry for type IV β-tubulin, a marker of respiratorycilia. 0=no cilia; 1=less than 30% cilia; 2=30% to 60% cilia; 3=greaterthan 60% cilia.

FIG. 13 is a series of images depicting that application of PHI with orwithout preservative does not affect murine septal ciliary beatfrequency. (A) continuous recording and analysis of ciliary beatfrequency from murine nasal septal explants demonstrates thatapplication of Solution 2 (0.075% potassium sorbate) (up arrow) does notalter ciliary beat frequency. Additionally, replacement of Solution 2with Locke Ringer's (down arrow) does not affect ciliary beat frequency.(B) normalization of ciliary beat frequency was performed to account forintrinsic differences in basal beat frequency of varying specimens. Noalteration in f_(PHI)/f_(basal) is evident (tracing between up and downarrow).

FIG. 14 is a series of images depicting that application of Solution 3with or without preservative does not affect murine septal ciliary beatfrequency. (A) continuous recording and analysis of ciliary beatfrequency from murine nasal septal explants demonstrates thatapplication of Solution 3 (0.075% potassium sorbate) (up arrow) does notalter ciliary beat frequency. Additionally, replacement of PHI withLocke Ringer's (down arrow) does not affect ciliary beat frequency. (B)normalization of ciliary beat frequency was performed to account forintrinsic differences in basal beat frequency of varying specimens. Noalteration in f_(PHI+MgBr)/f_(basal) is evident (tracing between up anddown arrow).

FIG. 15 is a series of images depicting Solution 2 and Solution 3accelerate mucosal regeneration with ciliated epithelium. Representativesections through the injury site stained with (A) hematoxylin and eosinor (B) immunohistochemistry for the respiratory ciliary marker, type IVβ-tubulin, demonstrate normal-appearing respiratory mucosa with a brushborder in the treated sinuses compared with the saline sinuses.

FIG. 16 is a series of graphs depicting the ciliation score byhematoxylin and eosin (H&E) and type IV β-tubulin immunohistochemistryanalysis for saline, Solution 2, and Solution 3. The graphs show abreakdown of ciliation score as a percentage of total observations foreach treatment by (A) H&E staining and (B) type IV β-tubulinimmunohistochemistry.

FIG. 17 is an image of a rabbit snout following mucosal stripping andsubsequent healing. A montage of multiple images reconstructing a rabbitsnout following surgery and treatment. Arrows illustrate the surgicalantrostomy created to access the sinus, and through which the irrigatingcatheter was placed. Dashed lines represent the approximate superior andinferior margin of the mucosal injury while the boxes represent thedistribution of photomicrographs obtained for analysis.

FIG. 18 is a series of images depicting a semi quantitative grading ofreciliation. (A) sections of rabbit snout fixed and stained withhematoxylin and eosin; (B) sections of rabbit snout fixed and subjectedto immunohistochemistry for type IV β-tubulin, a marker of respiratorycilia. 0=no cilia; 1=less than 30% cilia; 2=30%-60% cilia; 3=>60% cilia.

FIG. 19 is a series of images depicting that the application of Solution3 accelerates mucosal regeneration with ciliated epithelium.Representative sections through the injury site stained with (A)hematoxylin and eosin or (B) immunohistochemistry for the respiratoryciliary marker, type IV β-tubulin demonstrate normal-appearingrespiratory mucosa with a brush border in the treated sinuses comparedto the saline treated sinuses with sparse ciliation and flattenedfibroblastic-appearing cells.

FIG. 20 is an image depicting a representative section through theinjury site demonstrating the respiratory mucosa after treatment withsaline.

FIG. 21 is an image depicting a representative section through theinjury site demonstrating the respiratory mucosa after treatment withSolution 3 (sterile isotonic formulation containing K⁺, Rb⁺, Ca²⁺, Zn²⁺,Mg²⁺, and Br⁻ without the preservative potassium sorbate).

FIG. 22 is an image depicting a representative section through theinjury site demonstrating the respiratory mucosa after treatment withSolution 4 (sterile isotonic formulation containing Mg²⁺, Br⁻, Na+, Cl−,and SO₄ ²⁻ without the preservative potassium sorbate).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In the following detailed description of the preferred embodiments,reference is made to the accompanying drawings, which form a parthereof, and within which are shown by way of illustration specificembodiments by which the invention may be practiced. It is to beunderstood that other embodiments by which the invention may bepracticed. It is to be understood that other embodiments may be utilizedand structural changes may be made without departing from the scope ofthe invention.

DEFINITIONS

All numerical designations, such as pH, temperature, time,concentration, and molecular weight, including ranges, areapproximations which are varied up or down by increments of 1.0 or 0.1,as appropriate. It is to be understood, even if it is not alwaysexplicitly stated that all numerical designations are preceded by theterm “about”. It is also to be understood, even if it is not alwaysexplicitly stated, that the reagents described herein are merelyexemplary and that equivalents of such are known in the art and can besubstituted for the reagents explicitly stated herein.

Concentrations, amounts, solubilities, and other numerical data may beexpressed or presented herein in a range format. It is to be understoodthat such a range format is used merely for convenience and brevity andthus should be interpreted flexibly to include not only the numericalvalues explicitly recited as the limits of the range, but also toinclude all the individual numerical values or sub-ranges encompassedwithin that range as if each numerical value and sub-range is explicitlyrecited. As an illustration, a numerical range of “about 1 to about 5”should be interpreted to include not only the explicitly recited valuesof about 1 to about 5, but also include the individual values andsub-ranges within the indicated range, to the hundredth of the unitunless specifically specified otherwise. Thus, included in thisnumerical range are individual values such as 2, 3, and 4 and sub-rangessuch as from 1-3, from 2-4 and from 3-5, etc. This same principleapplies to ranges reciting only one numerical value. Furthermore, suchan interpretation should apply regardless of the range or thecharacteristics being described.

The term “about” as used herein is not intended to limit the scope ofthe invention but instead encompass the specified material, parameter orstep as well as those that do not materially affect the basic and novelcharacteristics of the invention. The term “about” or “approximately” asused herein refers to being within an acceptable error range for theparticular value as determined by one of ordinary skill in the art,which will depend in part on how the value is measured or determined,i.e. the limitations of the measurement system, i.e. the degree ofprecision required for a particular purpose, such as a pharmaceuticalformulation. For example, “about” can mean within 1 or more than 1standard deviation, per the practice in the art. Alternatively, “about”can mean a range of up to 20%, preferably up to 10%, more preferably upto 5% and more preferably still up to 1% of a given value.Alternatively, particularly with respect to biological systems orprocesses, the term can mean within an order of magnitude, preferablywithin 5-fold, and more preferably within 2-fold, of a value. Whereparticular values are described in the application and claims, unlessotherwise stated, the term “about” meaning within an acceptable errorrange for the particular value should be assumed.

The term “composition” as used herein describes an agent, compound,chemical, or extract that can be administered or tested by the presentinvention as promoting wound healing in a subject. The chemical can beof any composition such as inorganic, organic, or a biomolecule. Abiomolecule can be a molecule of any biological origin that can be foundin or produced by, at least in part, a cell and is meant to include ionssuch as the metal ions of the present invention. “Composition” is usedinterchangeably herein with “solution”, “compound”, “agent”, “chemical”,“ingredient” and “extract”.

The term “ion” as used herein describes a molecule, a portion of amolecule (e.g. Zwitterion), cluster of molecules, molecular complex,moiety or atom in which the total number of electrons is not equal tothe total number of protons thus giving it a net positive (cation) or anegative (anion) electronic charge. Alternatively, the molecule may notcontain a net positive or negative charge but can be made to contain anet positive or negative charge. Both cations and anions are included inthe term “ion”. Ions of the present invention may be produced bydissolving salts, including but not limited to, potassium citrate;magnesium chloride; rubidium chloride; calcium chloride; zinc chloride;magnesium bromide; and magnesium sulfate. Cations useful in the presentinvention include, but are not limited to, potassium, magnesium,rubidium, calcium, and zinc. Anions useful in the present inventioninclude, but are not limited to, citrate, chloride, bromide, andsulfate.

The term “counterion” as used herein describes an ion that accompaniesan ionic species in order to maintain electric neutrality in thecomposition. For example, the counterion of the sodium cation may be thechloride anion and vice versa.

The term “Solution 1” or “original solution” as used herein refers to asterile hypertonic formulation containing K⁺, Rb⁺, Ca²⁺, and Zn²⁺ withor without the preservative potassium sorbate.

The term “Solution 2” as used herein refers to a sterile isotonicformulation containing K⁺, Rb⁺, Ca²⁺, and Zn²⁺ without the preservativepotassium sorbate.

The term “Solution 3” as used herein refers to a sterile isotonicformulation containing K⁺, Rb⁺, Ca²⁺, Zn²⁺, Mg²⁺, SO₄ ²⁻, and Br⁻without the preservative potassium sorbate.

The term “Solution 4” as used herein refers to a sterile isotonicformulation containing Mg²⁺, Br⁻, Na+, Cl−, and SO₄ ²⁻ without thepreservative potassium sorbate.

“Subject” is used to describe an animal, preferably a human, to whomtreatment is administered, including prophylactic treatment with thecompositions of the present invention. “Subject” and “patient” are usedinterchangeably herein.

The term “therapeutically effective amount” as used herein is defined asconcentrations or amounts of components which are sufficient to effectbeneficial or desired clinical results, including, but not limited to,promoting wound healing; promoting debridement of tissue after surgery;reducing reactive oxygen species; modulating protease binding;modulating matrix metalloproteinase levels; promoting cilialregeneration, particularly in mucosal membranes; enhancing interactionwith enzymes involved in biosynthetic pathways necessary for woundhealing; reducing the level of superoxide anions associated with thewound; modulating human complement activation; reducing scar tissue;reducing inflammation; upregulating growth factors; reducing cytokines;and decreasing bacteria in the wound. Compositions of the presentinvention can be used to effect a favorable change in the conditionwhether that change is an improvement or a complete elimination of awound. In accordance with the present invention, a suitable single dosesize is a dose that is capable of preventing or alleviating (reducing oreliminating) a symptom in a subject when administered one or more timesover a suitable time period. One of skill in the art can readilydetermine appropriate single dose sizes for systemic administrationbased on the size of the animal and the route of administration. Theeffective amount of the compositions of the present inventionencompasses providing enhanced wound healing without causing significantside effects or adverse reactions. The terms “therapeutically effectiveamount”, “therapeutically effective dose”, “effective amount”, and“effective dose” are used interchangeably herein.

The term “ameliorates” as used herein refers to a change in state from afirst to a second state, where the first state is the condition of theepithelium prior to the application of the composition, and the secondstate is the condition of the epithelium after application of thecomposition where the condition of the second state is improved ascompared to the first state as a result of the application of thecomposition.

The term “ciliated epithelium” or “ciliated tissue” as used hereinrefers to all ciliated epithelial structures including, but not limitedto, nasal mucosa, sinus and paranasal sinus, trachea and middle ear.

The term “matrix metalloproteinase” or “MMP” as used herein refers toproteases which are zinc-dependent endopeptidases that are capable ofdegrading extracellular matrix proteins as well as process a number ofbioactive molecules. During wound repair, different MMPs can be producedby multiple cell types. For example, MMP-2 is only produced byinflammatory cells while MMP-9 is produced by keratinocytes as well asinflammatory cells. MMPs are not normally actively expressed inuninjured skin either in the epidermis or dermis. Reducing MMP levels inwounds can assist in enhancing wound healing.

The term “administrating” or “administration” as used herein are definedas the process by which the compositions of the present invention aredelivered to the individual for treatment or prevention purposes. Thecomposition can be delivered nasally, orally, rectally, percutaneously,intravaginally, topically, intraocularly, intrauterinely, or byinjection in dosage unit formulations containing conventional nontoxicpharmaceutically acceptable carriers, adjuvants, and vehicles asdesired. For example, in preparing the compositions in oral dosage form,any of the usual pharmaceutical media may be employed, such as, forexample, water, glycols, oils, alcohols and the like in the case of oralliquid preparations such as suspensions, syrups, elixirs and solutions;or solid carriers such as starches, sugars, kaolin, lubricants, binders,disintegrating agents and the like in the case of powders, pills,capsules and tablets. For injection compositions, the carrier willusually comprise sterile water, at least in large part, though otheringredients, for example, to aid solubility, may be included. Injectablesolutions, for example, may be prepared in which the carrier comprisessaline solution. In the compositions suitable for percutaneousadministration, the carrier optionally comprises a penetration enhancingagent and/or a suitable wettable agent, optionally combined withsuitable additives of any nature in minor proportions, which additivesdo not cause any significant deleterious effects on the skin. Saidadditives may facilitate the administration to the skin and/or may behelpful for preparing the desired compositions. These compositions maybe administered in various ways, e.g. as a transdermal patch, or aspot-on treatment.

In some embodiments, the interval between each administration isconstant. The administration of the composition may be spaced at timeintervals such as at about every 24 hours; at about every 23 hours; atabout every 22 hours; at about every 21 hours; at about every 20 hours;at about every 19 hours; at about every 18 hours; at about every 17hours; at about every 16 hours; at about every 15 hours; at about every14 hours; at about every 13 hours; at about every 12 hours; at aboutevery 10 hours; at about every 9 hours; at about every 8 hours; at aboutevery 7 hours; at about every 6 hours; at about every 5 hours; at aboutevery 4 hours; at about every 3 hours; at about every 2 hours; or atabout every 1 hour. Administration can also be continuous and adjustedto maintaining a level of the compound within any desired and specifiedrange.

The amount of ions in the composition will depend on absorption,distribution, metabolism, and excretion rates as well as other factorsknown to those of skill in the art. Dosage values may also vary with theseverity of the condition to be alleviated. The compounds may beadministered once, or may be divided and administered over intervals oftime. It is to be understood that administration may be adjustedaccording to individual need and professional judgment of a personadministrating or supervising the administration of the compositionsused in the present invention. The amount of ions in the solution ispresented as the concentration in percent (w/v). This is equivalent tothe mg of the particular ion per 100 mL of working solution.

The dose of the compounds administered to a subject may vary with theparticular composition, the method of administration, and the particulardisorder being treated. The dose should be sufficient to affect adesirable response, such as a therapeutic or prophylactic responseagainst a particular disorder or condition. For example, the dosage of acompound of the invention administered in the form of a nasal spray canbe about 0.1 oz. (equivalent to about 3 ml or 3 cc) to about 30 oz.(equivalent to about 882.2 ml or cc), including all units in between,when the composition is administered at least once per day. It iscontemplated that one of ordinary skill in the art can determine andadminister the appropriate dosage of compounds disclosed in the currentinvention according to the foregoing considerations.

The administration of the composition can be extended over an extendedperiod of time, such as from about a day or shorter up to about a yearor longer. For example, the dosing regimen can be extended over a periodof any of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, and 31 days orover a period of any of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15,18, 21 or 24 months or over a period of 1, 2, 3, 4, 5 or longer years.In an embodiment, the composition may be administered for about 14 days.In another embodiment, the composition may be administered for at least14 days. In a further embodiment, the composition may be administeredfor between about 14 days and about 30 days. In some embodiments, thereis no break in the dosing schedule. In some embodiments, the intervalbetween each administration is no more than about 24 hours.

The compounds used in the present invention may be administeredindividually, or in combination with or concurrently with one or moreother compounds known to enhance wound healing. Additionally, compoundsused in the present invention may be administered in combination with orconcurrently with other therapeutics for debridement of necrotic ordamaged tissue; therapeutics for reducing reactive oxygen species;therapeutics for modulating protease binding; and therapeutics formodulating matrix metalloproteinase levels.

The pharmaceutical compositions of the subject invention can beformulated according to known methods for preparing pharmaceuticallyuseful compositions. Furthermore, as used herein, the phrase“pharmaceutically acceptable carrier” means any of the standardpharmaceutically acceptable carriers. The pharmaceutically acceptablecarrier can include diluents, adjuvants, and vehicles, as well asimplant carriers, and inert, nontoxic solid or liquid fillers, diluents,or encapsulating material that does not react with the activeingredients of the invention. Examples include, but are not limited to,phosphate buffered saline, physiological saline, water, and emulsions,such as oil/water emulsions. The carrier can be a solvent or dispersingmedium containing, for example, saline, ethanol, polyol (for example,glycerol, propylene glycol, liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils. Formulations aredescribed in a number of sources that are well known and readilyavailable to those skilled in the art. For example, Remington'sPharmaceutical Sciences (Martin E W [1995] Easton Pa., Mack PublishingCompany, 19^(th) ed.) describes formulations which can be used inconnection with the subject invention.

The preparation process of the composition is designed to maintain themaximum therapeutic effect of the composition and method. The parametersof the composition are controlled such as its isotonicity, pH range, andtemperature. For example, the pH of the solution of the presentinvention may be slightly acidic to limit any potential stinging uponuse and to encourage protease inhibition since protease inhibition ismaximized at an acidic pH. The term “slightly acidic” refers to a pHthat ranges from about 5 to about 6.9. The composition of the presentinvention may be an isotonic solution or a hypertonic solution. Tonicityfor the solution may be between about 0.9% and about 4%. In a beneficialembodiment, the solution is isotonic or slightly hypertonic.

The present invention can be applied to enhance wound healing, bothinternally and externally. Administration of the composition of thepresent invention provides an improvement in wound healing for all typesof epithelium and mucosa including, but not limited to, the respiratorysystem including, but not limited to, the throat, trachea, lungs, nasalcavity, sinuses, and larynx; the middle ear; the gastrointestinal systemincluding, but not limited to the esophagus, the stomach, and the smalland large intestines; the reproductive system including, but not limitedto the vagina, cervix, uterus, fallopian tubes and ovaries; the dermis;and the eyes.

While the present invention can be applied to both internal and externalwound healing as well as to tissue health in general, it will bedescribed in connection with chronic rhinosinusitis, maxillary mucosalinjury, and sinonasal inflammation in particular. The description withrelation to chronic rhinosinusitis is used as an example and is not tobe construed as limiting as the process of the present inventionincludes treatment of any mucosal tissue. In a representativeembodiment, the mucosal membranes of the nasal passages are treated witha novel, specifically formulated mineral cationic solution thatcleanses, debrides, soothes and heals inflamed and injured sinonasalciliated mucosa.

In the chronic rhinosinusitis example, the solution may be applied tothe nasal passages and paranasal sinuses by a number of forms including,but not limited to, nasal drops; nasal sprays; nasal mists; nasal rinse;nebulizers; creams; and gels.

The solution of the present invention can be buffered with a weak acidincluding, but not limited to, citric acid, acetic acid, lactic acid andgluconic acid in a pH range between about 5.0 to about 7.0 including allunits in between. In an embodiment, the pH range can be between about6.0 to about 6.8. In a further embodiment, the pH can be about 6.5.Citrate (C₆H₈O₇ ³⁻) may be used to adjust the pH in some embodiments.

In an embodiment, the solution can include citric acid (C₆H₈O₇) in therange of about 0.01-0.1% (w/v), including all units in between to thehundredth of a percent. In an embodiment, the range may be between about0.018-0.082% (w/v) including all units in between to the thousandth of apercent. In a further embodiment, the concentration may be about 0.03%(w/v). In a further embodiment, the concentration may be about 0.03654%(w/v). The pH of the final solution can be adjusted by the addition of abase including, but not limited to, ammonia, carbonates and hydroxidesof barium, calcium, cesium, lithium, potassium, rubidium, sodium, andstrontium. In a beneficial embodiment, the base is potassium hydroxide(KOH).

A listing of potential ions as well as ranges of concentrations that canbe used in Solutions 1-4 are given below. The type and percentage of ionused varies between the solutions. Not all ions are present in allsolutions. The cations may be obtained using different salts other thanthose listed below. These alternative salts may have different hydrationstates or use different counterions. Examples of salts that may be usedinclude, but are not limited to, citrate, chloride, bromide, sulfate,bicarbonate, lactate, and phosphate. One of ordinary skill in the artwould recognize the alternative salts that may be used in conjunctionwith the present invention.

Potassium citrate, typically K₃citrate.H₂O, is present in thecomposition such that the potassium concentration [K⁺] is in the rangeof about 0.02-3.0% (w/v), including all units in between to thehundredth of a percent. In an embodiment, the range may be between about0.2056-1.2336% (w/v) including all units in between to the tenthousandth of a percent. In a further embodiment, the concentration maybe about 0.4% (w/v). In a further embodiment, the concentration may beabout 0.4112% (w/v). The citrate concentration [C₆H₅O₇ ³⁻] can be in therange of about 0.03-4.7% (w/v), including all units in between to thehundredth of a percent. In an embodiment, the range may be between about0.33145-1.9887% (w/v) including all units in between to the hundredthousandth of a percent. In a further embodiment, the concentration maybe about 0.6% (w/v). In a further embodiment, the concentration may beabout 0.6629% (w/v).

Magnesium chloride, typically MgCl₂.6H₂O, is present in the compositionsuch that the magnesium concentration [Mg²⁺] is in the range of about0.007-1.14% (w/v), including all units in between to the thousandth of apercent. In an embodiment, the range may be between about 0.072-0.43182%(w/v), including all units in between to the hundred thousandth of apercent. In a further embodiment, the concentration may be about 0.14%(w/v). In a further embodiment, the concentration may be about 0.14394%(w/v). The chloride [Cl⁻] concentration can be in the range of about0.001-2.3% (w/v) including all units in between to the thousandth of apercent. In an embodiment, the range may be between about 0.21-1.255%(w/v) including all units in between to the thousandth of a percent. Ina further embodiment, the concentration may be about 0.41% (w/v). In afurther embodiment, the concentration may be about 0.41832% (w/v).

Rubidium chloride, typically RbCl, is present in the composition suchthat the rubidium concentration [Rb⁺] is in the range of about0.0001-4.0% (w/v), including all units in between to the ten thousandthof a percent. The lower end of this range may be 0 in cases whererubidium is not present in the solution. In an embodiment, the range maybe between about 0.0015-0.0095% (w/v) including all units in between tothe ten thousandth of a percent. In a further embodiment, theconcentration may be about 0.003% (w/v). In a further embodiment, theconcentration may be about 0.0031453% (w/v).

Calcium chloride, typically CaCl₂.2H₂O, is present in the compositionsuch that the calcium concentration [Ca²⁺] is in the range of about0.000005-1.3% (w/v), including all units in between to the millionth ofa percent. The lower end of this range may be 0 in cases where calciumis not present in the solution. In an embodiment, the range may bebetween about 0.000057-0.00034284% (w/v) including all units in betweento the millionth of a percent. In a further embodiment, theconcentration may be about 0.00011428% (w/v).

Zinc chloride, typically ZnCl₂, is present in the composition such thatthe zinc concentration [Zn²⁺] is in the range of about 0.0000057-2.12%(w/v), including all units in between to the millionth of a percent. Thelower end of this range may be 0 in cases where zinc is not present inthe solution. In an embodiment, the range may be between about0.000012-0.0000741% (w/v). In a further embodiment, the concentrationmay be about 0.0000247% (w/v).

Magnesium bromide, typically MgBr₂.6H₂O, is present in the compositionsuch that the bromide concentration [Br⁻] is in the range of about0.0002-0.2.6% (w/v), including all units in between to the tenthousandth of a percent. The lower end of this range may be 0 in caseswhere bromide is not present in the solution. In an embodiment, therange may be between about 0.0022-0.01335516% (w/v). In a furtherembodiment, the concentration may be about 0.00445172% (w/v).

Magnesium sulfate, typically MgSO⁴.7H₂O, is present in the compositionsuch that the sulfate concentration [SO₄ ²⁻] is in the range of about0.000078-4.5% (w/v), including all units in between to the millionth ofa percent. The lower end of this range may be 0 in cases where sulfateis not present in the solution. In an embodiment, the range may bebetween about 0.00077-0.004677% (w/v) including all units in between tothe millionth of a percent. In a further embodiment, the concentrationmay be about 0.001559% (w/v).

Sodium chloride, typically NaCl, is present in the composition such thatthe sodium concentration [Na⁺] is in the range of about 0.005%-3.0%(w/v) including all units in between to the thousandth of a percent. Thelower end of this range may be 0 in cases where sodium is not present inthe solution. In one embodiment, [Na⁺] is in the range of about0.05%-0.4833% (w/v) including all units in between to the ten thousandthof a percent. In a further embodiment, the concentration may be about0.16% (w/v). In another embodiment, [Na⁺] concentration is about0.1610936% (w/v).

Before use, the final solution can be sterilized using any one of anumber of methods, including, but not limited to, heating at 80° C. for2 hours, sterile filtering through an about 0.2 m filter, gammairradiation, or any other appropriate method known to those skilled inthe art.

Various counterions may be used in the solutions of the presentinvention including, but not limited to, citrate, chloride, bromide,sulfate, hydroxide, bicarbonate, carbonate, phosphate, lactate,glutamate, and acetate as well as other anions that are found inphysiological systems. In some embodiments, non-physiological anionssuch as bromide can be used.

The mineral salt constituents of the solutions can include, but are notlimited to: potassium citrate, magnesium chloride, rubidium chloride,calcium chloride, zinc chloride, magnesium bromide, magnesium sulfate,and sodium chloride. A pharmaceutically acceptable carrier including,but not limited to, water and/or aqueous solutions of water andhydrophilic polymers such as polyethylene glycol and methyl celluloseare added to each solution to act as a debrider and moisturizer.

The composition can contain additional inactive ingredients including,but not limited to, preservatives such as potassium sorbate, benzoicacid, chlorhexidine acetate and/or gluconate, parabens and potassiumsorbate as well as emulsifying agents and stabilizers such as glycerolmonosterate and methylcellulose, respectively.

The composition of the present invention can be administered in avariety of ways to a subject as described above. For example, insinonasal administration the easiest route of administration is througha liquid in the form of a nasal spray, nasal drops, or nasal mist. Ifadministered by nasal spray, about 0.1 oz. to about 30 oz. may beadministered to the subject at least once per day. In a beneficialembodiment, the nasal spray is administered about twice per day. As anon-limiting example, the nasal spray may be administered in a dosage of8 ounces, twice a day, with 4 ounces being administered into eachnostril twice a day. The nasal spray may be administered between about 1day and about 14 days or longer. In a beneficial embodiment, the nasalspray is administered for about 14 days.

Specific Solution Formulations

Solution 2 (sterile isotonic formulation containing K⁺, Rb⁺, Ca²⁺, andZn²⁺ without the preservative potassium sorbate), may contain frombetween about 0.02-3.0% (w/v) of K⁺; from between about 0.0001-4.0%(w/v) of Rb⁺; from between about 0.000005-1.3% (w/v) of Ca²⁺; and frombetween about 0.0000057-2.12% (w/v) of Zn²⁺. A beneficial amount of K⁺is about 0.924% (w/v). A beneficial amount of Rb⁺ is about 0.007068%(w/v). A beneficial amount of Ca²⁺ is about 0.0002568% (w/v). Abeneficial amount of Zn²⁺ is about 0.0000554% (w/v). Solution 2 may alsocontain citrate (C₆H₅O₇ ³⁻) in a range from between about 0.03-4.7%(w/v). A beneficial amount of C₆H₅O₇ ³⁻ is about 1.57% (w/v). Solution 2may also contain citric acid (C₆H₅O₇) in a range from between about0.01-0.1% (w/v). If chloride salts are used, Cl− can be present in about0.0034464% (w/v) in a beneficial embodiment. In one beneficialembodiment, Solution 2 may be administered at least once per day.

Solution 3 (sterile isotonic formulation containing K⁺, Rb⁺, Ca²⁺, Zn²⁺,Mg²⁺, and Br⁻ without the preservative potassium sorbate), may containfrom between about 0.02-3.0% (w/v) of K⁺; from between about 0.0001-4.0%(w/v) of Rb⁺; from between about 0.000005-1.3% (w/v) of Ca²⁺; frombetween about 0.0000057-2.12% (w/v) of Zn²⁺; from about 0.007-1.14%(w/v) of Mg²⁺; and from about 0.0002-2.6% (w/v) of Br⁻. A beneficialamount of K⁺ is about 0.4112% (w/v). A beneficial amount of Rb⁺ is about0.0031453% (w/v). A beneficial amount of Ca²⁺ is about 0.00011428%(w/v). A beneficial amount of Zn²⁺ is about 0.0000247% (w/v). Abeneficial amount of Mg²⁺ is about 0.14394% (w/v). A beneficial amountof Br⁻ is about 0.00445172% (w/v). Solution 3 may also contain citrate(C₆H₅O₇ ³⁻) in a range from between about 0.03-4.7% (w/v) and sulfate(SO₄ ²⁻) in a range from about 0.000078-4.5% (w/v). A beneficial amountof C₆H₅O₇ ³⁻ is about 0.6629% (w/v). A beneficial amount of SO₄ ²⁻ isabout 0.001559% (w/v). Solution 3 may also contain citric acid (C₆H₅O₇)in a range from between about 0.01-0.1% (w/v). If chloride salts areused, Cl− can be present in about 0.41832% (w/v) in a beneficialembodiment. The amount of Cl− can be adjusted to correspond to theconcentration of other ions present in the solution to ensure that atonicity of between about 0.9% and about 3.5% is maintained in thesolution. In one beneficial embodiment, Solution 3 may be administeredat least once per day.

In one beneficial embodiment of Solution 3, the following salts andconcentrations may be used in preparing 10 L of solution: potassiumcitrate at a concentration of about 1.13728% (w/v); magnesium chlorideat a concentration of about 1.195% (w/v); rubidium chloride at aconcentration of about 0.00445% (w/v); calcium chloride at aconcentration of about 0.0004192% (w/v); zinc chloride at aconcentration of about 0.0000514% (w/v); magnesium bromide at aconcentration of about 0.00814% (w/v); magnesium sulfate at aconcentration of about 0.004% (w/v); and citric acid at a concentrationof about 0.03654% (w/v). Water is added to the solution and the pH ispreferably adjusted to about 6.5. Less than about 2.4% (w/v) of thesalts are contained in the solution. The amounts disclosed are theamounts that can be present in one 6 oz. (equivalent to about 177.44 ml)bottle of solution.

Solution 4 (sterile isotonic formulation containing Mg²⁺, Br⁻, SO₄ ²⁻,Na⁺, and Cl− without the preservative potassium sorbate), may containfrom between about 0.007-1.14% (w/v) of Mg²⁺; from between about0.0002-2.6% (w/v) of Br⁻; from between about 0.000078-4.5% (w/v) of SO₄²⁻; from between about 0.005-3.0% (w/v) of Na⁺; and from between about0.001-2.3% (w/v) of Cl−. A beneficial amount of Mg²⁺ is about 0.14394%(w/v). A beneficial amount of Br⁻ is about 0.00445172% (w/v). Abeneficial amount of SO₄ ²⁻ is about 0.001559% (w/v). A beneficialamount of Na⁺ is about 0.1610936% (w/v). A beneficial amount of Cl− isabout 0.6652122% (w/v). In one beneficial embodiment, Solution 4 may beadministered at least once per day.

In an embodiment of Solution 4, the amounts of salts and concentrationsof salts can be obtained similarly to the formulation of Solution 3 byusing the concentration of ions disclosed above for Solution 4 andadjusting for the presence of the corresponding salt.

The solutions of the present invention are preferably prepared inbatches of at least 10 L due to the small amount needed of some of theingredients as well as for convenience. All ingredients are used belowtheir solubility limits and thus they are all readily soluble in theaqueous medium of the solution. Constant stirring is generally neededduring preparing the solutions. While the ingredients may be added inany order, it may be preferable to add the ingredients that are presentin larger quantities first in order to aid in the mixing process. Someingredients may need to be prepared in dilute “pre-solutions” due toconsiderations of accurate weighing of the lesser amounts ofingredients.

Preliminary Studies on Biocompatibility and Safety of the PresentInvention Cytotoxicity

Following the requirements of International Organization forStandardization (ISO 10993-5): Biological Evaluation of MedicalDevices—Part 5: Tests for Cytotoxicity, In Vitro Methods, the presentinvention showed no evidence of causing any cell lysis or toxicity.L-929 mouse fibroblast cells were seeded in 10 cm₂ wells and incubatedat 37° C. in the presence of 5% CO₂ to obtain subconfluent monolayers ofcells. When the cells had grown to subconfluency, the growth medium wasremoved from over them and replaced with 2 mL of 2% agarose in 2×MEM(Minimal Essential Medium supplemented with 10% fetal bovine serum, 4%antibiotics (200 units/mL penicillin, 200 g/mL streptomycin, and 5.0g/mL amphotericin) and 2% (4 mM) L-glutamine) and neutral red. Theagarose mixture was allowed to solidify over the cells to form anagarose overlay. The agarose over the cells was covered with fourarticles in triplicate as follows:

1) 0.1 mL sodium chloride (0.9%) on a filter disc as a negative control2) A 1 cm length of HDPE as a negative control3) A 1 cm×1 cm portion of latex as a positive control4) 0.1 mL present invention on a filter disc

These articles were incubated on the cells at 37° C. in the presence of5% CO₂ for 24 hours. After incubation, the cells were examinedmacroscopically for cell decolorization around the test article andcontrols to determine the zone of cell lysis (if any). After macroscopicexamination, the cell monolayers were examined microscopically (100×) toverify any decolorized zones and to determine cell morphology inproximity to the article. Scoring for cytotoxicity was based on thefollowing criteria in Table 1 below:

TABLE 1 Grade Reactivity Condition of Cultures 0 None No detectable zonearound or under specimen 1 Slight Some malformed or degenerated cellsunder specimen 2 Mild Zone limited to area under specimen 3 ModerateZone extending specimen size up to 1.0 cm 4 Severe Zone extendingfarther than 1.0 cm beyond specimen

For the suitability of the system to be confirmed, the negative controlmust have been a grade of 0 (reactivity none) and the positive controlmust have been a grade equal to or greater then a grade of 3 (reactivitymoderate to severe). The test article containing the preferredembodiment of the present invention passed the test if all threemonolayers were less than or equal to a grade of 2 (reactivity mild).The scores obtained were as shown in Table 2 below:

TABLE 2 Zone of Articles Lysis Grade Reactivity Test article filterdiscs (1) 0 0 None containing extract of (2) 0 0 None the presentinvention (3) 0 0 None Filter disc containing (1) 0 0 None 0.9% normalsaline (2) 0 0 None (3) 0 0 None Negative Control: (1) 0 0 None 1 cmlength of HDPE (2) 0 0 None (3) 0 0 None Positive Control: (1)* 7 3Moderate 1 cm × 1 cm portion (2)* 7 3 Moderate of latex (3)* 8 3Moderate *Complete cell lysis was beneath the sample

Conclusion: The present invention showed no evidence of causing any celllysis or toxicity. The safety of the present invention was confirmed inthe second biocompatibility test as follows:

Intracutaneous Reactivity

Following the requirements of International Organization forStandardization (ISO 10993-10): Biological Evaluation of MedicalDevices—Part 10: Tests for Irritation and Delayed-Type Hypersensitivity(modified for a chemical solution), the present invention showed noerythema and no edema when injected intracutaneously into rabbits.

Two male New Zealand White rabbits were used for the study. Fur wasclipped off the back of both animals over an area just large enough forthe injections, and 0.2 mL dose of Solution 3 (test article) wasinjected by the intracutaneous route into five separate sites on theright side of the back of each animal. Similarly, 0.2 mL of 0.9% sodiumchloride (USP solution) was injected into five sites on the left side ofthe back of each animal as a negative control. Injections were placedapproximately 2 cm apart. The appearance of each injection site wasnoted immediately after injection and at 24, 48, and 72 hours afterinjection. Any reactions were scored on a 0 to 4 basis and any reactionsat the injection site were also noted. The reactions were evaluatedaccording to the subjective rating scale depicted in Table 3 below:

TABLE 3 Score Erythema Edema 0 No erythema No edema 1 Very slighterythema Very slight edema (barely perceptible) (barely perceptible) 2Well-defined erythema Well-defined edema (edges of area Well-defined bydefinite rising) 3 Moderate erythema Moderate edema (raisedapproximately 1 mm) 4 Severe erythema (beet redness) Severe edema(raised more to eschar Formation than 1 mm, and Extending preventinggrading of erythema beyond exposure area)

The mean erythema and edema scores for both the control and test articlesites in each animal at each scoring interval were calculated asfollows: All mean erythema and edema scores for the test and controlarticles were totaled and divided by 12 (2 animals×3 grading periods×2grading categories) to determine to overall mean scores for eacharticle. The difference between to overall mean score of the controlarticle subtracted from the overall mean score of the test article wasdetermined. If the overall mean score of the test article was less thanthe overall mean score of the control article, 0.0 was reported. Therequirements of the test were met if the difference between the overalltest article mean score and overall control article mean score was 1.0or less. Both animals appeared normal throughout the study and theresults of all scores are shown in Table 4 below:

TABLE 4 Scoring Interval Body 24 hours 48 hours 72 hours Weight TestControl Test Control Test Control Animal Number Sex (kg) ER ED ER ED ERED ER ED ER ED ER ED 69695 Male 2.4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00 0 0 0 0 0 Mean Score 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.069698 Male 2.4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Mean Score0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Conclusion: The present invention injected intracutaneously into rabbitsresulted in no erythema and no edema.

Systemic Toxicity

The Systemic Toxicity Test was performed in accordance with therequirements of International Organization for Standardization (ISO10993-11): Biological Evaluation of Medical Devices—Part 11: Tests forSystemic Toxicity (modified to increase the observation period from 72hours to 5 days and to dose a solution instead of an extract), and basedon the United States Pharmacopeia, National Formulary, General Chapter<88>, Biological Reactivity Tests, In Vitro. The study was conducted inaccordance with the provisions of the FDA Good Laboratory Practice (GLP)Regulations (21 CFR, Part 58) evaluating Solution 3 (test article) foracute systemic toxicity in mice. Following the injection of a single 25mL/kg dose via the intraperitoneal route into mice, Solution 3 showed nomortality or evidence of systemic toxicity.

Ten Hla:(ICR)CVF male mice, approximately 4 weeks old and weighing 18-22grams at injection were divided into two groups to receive eitherSolution 3 (test article) or 0.9% sodium chloride (control article).Mice have historically been used to evaluate potential toxicity of testarticles. Each animal was injected via the intraperitoneal route with adose of 25 mL/kg of either test article or control article. The test andcontrol animals were observed for adverse reactions immediately afterdosing and at 1 and 4 hours after injection and daily for 7 days. Theanimals were also weighed daily for 7 days after dosing. If during theobservation period, none of the animals treated exhibited asignificantly greater reaction than the control animals, the testarticle met the test requirements. If two or more animals died, or ifabnormal behavior such as convulsions or prostration occurred in two ormore animals, or if body weight loss greater than 2 grams occurred inthree or more animals, the test article did not meet the testrequirements.

Results of the tests are shown in Tables 5 and 6 below:

TABLE 5 Mortality and Body Weight Data: Animal Weight (g) #Dead/ NumberDay 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 #Tested Test Article 6621 22 23 24 25 27 28 29 0/5 67 21 22 24 24 26 26 27 29 68 21 22 24 25 2627 28 29 69 22 23 24 26 27 28 29 30 70 18 19 20 20 22 23 23 24 ControlArticle 61 20 21 22 23 24 25 26 26 0/5 62 21 22 23 24 25 27 28 29 63 1820 21 22 24 25 26 27 64 20 20 22 23 24 26 26 27 65 19 21 22 23 24 25 2627

TABLE 6 Clinical Observations: Animal Number 66 61 67, 68, 69, 70 62,63, 64, 65 Time of Test Control Test Control Observation Article ArticleArticle Article Immediate Normal Normal Normal Normal 1 hour NormalNormal 4 hour Normal Normal Normal Normal Day 1 Normal Normal NormalNormal Day 2 Normal Normal Normal Normal Day 3 Normal Normal NormalNormal Day 4 Normal Normal Normal Normal Day 5 Normal Normal NormalNormal Day 6 Normal Normal Normal Normal Day 7 Normal Normal NormalNormal

As evidenced above, there was no mortality or evidence of systemictoxicity from the present invention injected into mice.

Dermal Contact Sensitization

The Dermal Contact Sensitization Test was performed in accordance withthe requirements of International Organization for Standardization (ISO10993-10): Biological Evaluation of Medical Devices—Part 10: Tests forIrritation and Delayed-Type Hypersensitivity. The study was conducted inaccordance with the provisions of the FDA Good Laboratory Practice (GLP)Regulations (21 CFR, Part 58) evaluating Solution 3 (test article) fordelayed dermal contact sensitization in a guinea pig maximization test.Following intradermal injection and occlusive patching of the test andcontrol articles, Solution 3 showed no evidence of causing delayeddermal contact sensitization in the guinea pig. Dose determinationsprior to testing established that the test article would be used at 100%concentration. Ten HlaHA)CVF Hartley albino guinea pigs were injectedwith the test article and five guinea pigs (same strain) were injectedwith the control article (0.9% Sodium Chloride). The Hartley albinoguinea pig has been used historically for sensitization studies. Theguinea pig is believed to be the most sensitive animal model for thistype of study. The fur over the dorsoscapular region was removed andthree rows of intradermal injections (two injections per row) were givento each animal within an approximate 2 cm×4 cm boundary of the furclipped area as illustrated below.

Induction 1: Control Animals:

a. 0.1 mL of 50/50 (v/v) mixture of Freund's Complete Adjuvant (FCA) andthe control articleb. 0.1 mL of the control articlec. 0.1 mL of a 1:1 mixture of the 50/50 (v/v) control article/FCAmixture and the control article

Test Animals:

a. 0.1 mL of a 50/50 (v/v) mixture of Freunds Complete Adjuvant (FCA)and the control articleb. 0.1 mL of the test articlec. 0.1 mL of a 1:1 mixture of the 50/50 (v/v) control article/FCAmixture and the test article

To minimize tissue sloughing the “a” and “c” injections were slightlydeeper than the “b”. The site “c” injection was administered slightlymore caudal than site “b”.

Induction 2:

At 6 days (±1 day) after completion of the ‘Induction 1 injection’, thefur over the dorsoscapular region (same area as used during Induction 1)of each animal was removed with an electric clipper. The area wastreated with a 10% (w/v) Sodium Lauryl Sulfate (SLS) suspension inpetroleum sufficient to coat the skin. The SLS suspension, applied toprovoke a mild acute inflammation, was massaged into the skin over theinjection site. The area was left uncovered. At 24 hours (±2 hours), anyremaining SLS residue was gently removed with a gauze pad. Anapproximate 2 cm×4 cm section of filter paper, saturated with 0.3 mL oftest article, was then topically applied to the previously injectedsites of the test animals. The control animals were similarly patchedwith the control article. Each patch was secured with a non-reactivetape and the trunk of each animal was wrapped with an elastic bandage.At 48 hours, the bandages and patches were removed.

Challenge:

At 14 days (±1 day) after completion of Induction 2, the fur was removedfrom the sides and flank areas with an electric clipper. Non-wovencotton disks containing in a Hill Top Chamber were saturated with 0.3 mLof the test article or the control article. The test article was appliedto the right flank of each animal and the control article was applied tothe left flank of each animal. The trunk of each animal was wrapped withan elastic bandage to maintain well occluded sites. At 24 hours, thewraps and Hill Top Chambers were removed. Any residue remaining at thesites was removed. Observations for dermal reactions were conducted at24 and 48 hours after challenge patch removal. Prior to each scoringprocedure, the sites were wiped with 35% isopropyl alcohol. Ifnecessary, the fur was removed from each site to facilitate scoring.Dermal reactions were scored in accordance with the criteria shown belowin Table 7:

TABLE 7 Patch Test Reaction Grading Scale No visible change 0 Discreteor patchy erythema 1 Moderate and confluent erythema 2 Intense erythemaand swelling 3

Animals were observed daily for general health and body weights wererecorded at pretreatment. Individual results of dermal scoring for thechallenge phase are provided in Table 8 below. Also provided are theresults of the last periodic positive control study using1-chloro-2,4-dinitrobenzene (DNCB) as positive control.

TABLE 8 TEST ARTICLE OF PRESENT INVENTION Dermal Reactions 24 Hour Score48 Hour Score Treatment Animal Control Test Control Test Group NumberSite Site Site Site Test 2600 Removed from study for humane reasons 26010 0 0 0 2602 0 0 0 0 2603 0 0 0 0 2604 0 0 0 0 2935 0 0 0 0 2937 0 0 0 02944 0 0 0 0 2954 0 0 0 0 2959 0 0 0 0 Negative 2126 0 0 0 0 Control2129 0 0 0 0 2130 0 0 0 0 2131 0 0 0 0 2928 0 0 0 0 LAST PERIODICPOSITIVE CONTROL - PERFORMED AUG. 14, 2010 Positive 22 0 1 0 1 Control23 0 1 0 1 Using 74 0 1 0 1 DNCB 257 0 2 0 2 407 0 1 0 1 410 0 1 0 1 4140 1 0 2 417 0 1 0 2 426 0 2 0 2 428 0 2 0 2 Negative 19 0 0 0 0 Control21 0 0 0 0 122 0 0 0 0 135 0 0 0 0 187 0 0 0 0

Conclusion: The known sensitizer, DCNB produced evidence of causingdelayed dermal contact sensitization in the Hartley strain of guinea pigconfirming the methodology as valid. The present invention showed noevidence of causing delayed dermal contact sensitization in the guineapig. ISO 10993-10 indicates that a minimum of 10 test animals shall betreated with each test sample and a minimum of five animals shall act asa solvent control group. On day 2 of the study, animal 2600 was removedfrom the study for humane reasons. This resulted in nine test animalsremaining in the test group. All nine test animals had scores of “0” atboth 24 hours and 48 hours, which was identical to the scores of thecontrol animals. Due to the consistency of the data for the testanimals, results from nine animals were considered sufficient to drawvalid conclusions and thus did not have any impact on testing.

Oral Toxicity

The Oral Toxicity Test was performed in accordance with the guidelinesof the Federal Hazardous Substances Act (FHSA) Regulations, 16 CFR 1500.There was no mortality or significant evidence of toxicity observed inthe test animals (rats). Solution 3 (test article) would not beconsidered toxic at a dose of 5 g/kg by the oral route in the rat. TenHla:(SD)CVF, five male and five female rats, weighing 215-243 grams atdosing were dosed by gavage (the oral route) with the preferredembodiment of the present invention (test article). The rat hashistorically been used to establish hazardous substance labeling data.The oral route of dosing is selected as the strongest challenge formaterials that could be accidentally ingested. The food for each rat wasremoved from each cage 16-20 hours prior to dosing. Each rat was weighedand gavaged with the test article via a stainless steel blunt-tippedcannula at a dose of 5 g/kg of body weight. The animals were thenreturned to their cages and food was returned after treatment. Animalswere observed immediately after dosing, at 4 hours, and daily for up to14 days for signs of illness or mortality. Body weights were recorded atdosing and at 14 days. At termination of the study, animals wereeuthanized and their viscera inspected by macroscopic examination. Basedon the FHSA Regulations, a substance is considered “toxic” if itproduces death within 14 days in 50% or more of a group of rats dosedwith a single 50 mg/kg to 5 g/kg dose. All animals were clinicallynormal throughout the 14 day study, no animals died during the 14 daystudy, body weight data were acceptable, and there were no macroscopicchanges in the viscera at necropsy that could be attributed to thesingle oral dose. Individual observations appear in Table 9 below:

TABLE 9 Clinical Animal Body Weight (g) Observations Necropsy Number SexDay 0 Day 14 (Days 0-14) Day 14) 7664 Male 240 337 7665 Male 243 356Normal Macroscopically Normal 7666 Male 236 324 Normal MacroscopicallyNormal 7667 Male 243 339 Normal Macroscopically Normal 7668 Male 243 355Normal Macroscopically Normal 7671 Female 215 242 Normal MacroscopicallyNormal 7672 Female 226 255 Normal Macroscopically Normal 7673 Female 222249 Normal Macroscopically Normal 7674 Female 228 262 NormalMacroscopically Normal 7675 Female 220 247 Normal Macroscopically NormalMean: 232 297 Normal Macroscopically Normal

All of the above safety studies (cytotoxicity, intracutaneousreactivity, systemic toxicity, dermal contact sensitization and oraltoxicity) were conducted with Solution 3 (isotonic formulation of K⁺,Rb⁺, Ca²⁺, Zn²⁺ Mg²⁺, Br⁻±potassium sorbate as preservative) as the testarticle. Since this solution contained the ions present in Solution 2and Solution 4, one of ordinary skill in the art could conclude that thesafety study results similarly apply to these solutions as well.

Ciliotoxicity

Mouse septal explants were harvested and placed in a glass perfusionchamber on a thermostatically controlled stage. The chamber was filedwith Locke Ringer's solution (136 mM NaCl, 5.6 mM KCl, 10 mM HEPES, 14.3mM NaHCO3, 1.2 mM MgCl2, 2.2 mM CaCl2 and 11.5 mM dextrose, pH 7.35) andthe temperature maintained between 35.5° C. and 37° C. Images werevisualized microscopically using a water immersion 63× objective anddifferential interference contrast optics. Images were captured using ahigh-speed monochromatic digital video camera at a sampling rate of 100frames per second and analyzed using virtual instrumentation softwarehighly customized to perform cilia beating frequency (CBF) analysis.Recordings were made every 30 seconds for 10 minutes. Once a stablebaseline of CBF was obtained (5-10 min) the Locke Ringer's solution wasreplaced by solutions containing various compositions of metal ionswhile maintaining visualization of the beating cilia. Recordingscontinued for 10 min. In such fashion ciliotoxicity was determined. Theformulations of the present invention that were tested in sterilesolutions included:

A. Isotonic formulation of K⁺, Rb⁺, Ca²⁺, Zn²⁺±potassium sorbate aspreservative (Solution 2)B. Isotonic formulation of K⁺, Rb⁺, Ca²⁺, Zn²⁺ Mg²⁺, Br⁻±potassiumsorbate as preservative (Solution 3)C. Hypertonic formulation of K⁺, Rb⁺, Ca²⁺, Zn²⁺ Mg²⁺, Br⁻±potassiumsorbate as preservative (Solution 1+Mg²⁺, Br⁻)

The tracings in FIG. 1 indicate that none of the formulations of thepresent invention produced any acute ciliotoxicity in the mouse explantsmodel. On the left hand side of each tracing is the cilial beatingfrequency in Locke Ringer's solution and after the break in each trace,the cilial beating frequency of the cells immersed in the indicatedformulation of the present invention are represented.

Example 1 Efficacy for the Treatment of Sinonasal Injury

To evaluate efficacy, a live rabbit model of maxillary sinus injury wasused to test the ability of the present invention to causere-epithelialization of the rabbit maxillary sinus mucosa after surgicaltrauma.

Pasteurella-free New Zealand white rabbits underwent maxillary sinussurgery. In the first experiment, surgery was performed to completelyremove the left maxillary sinus mucosa while only the mucosa of themedial wall was removed on the right side. After mucosal stripping, theperiosteal flaps were repositioned and the skin incision was closed withsutures. Rabbits were sacrificed at 2, 7, 15, and 22 days post surgeryand the snouts sectioned and processed for hematoxylin and eosinstaining for analysis of mucosal regeneration. A qualitative grading ofthe epithelia was performed. Twelve symmetrically distributed points ofeach maxillary sinus were evaluated by light microscopy. The epitheliumfound was classified as normal respiratory, transitional or squamousepithelium.

Results: No Treatment:

Two days after the sinus mucosal stripping procedure a medial ulcerationwas seen in the right maxillary sinus, while an almost complete absenceof epithelium was evidenced on the left sinus. The walls were covered bya fibrinous matrix with numerous red blood cells, platelets andinflammatory cells. Areas of denuded bone were also seen (FIG. 2).

Seven days after the mucosal stripping procedure, ulcerated areas wereevident, covered by transitional squamous epithelium, however, patchyareas of ulceration still existed (FIG. 3). By the end of the secondweek bone formation was observed on the left side. The anterior portionof the left maxillary sinus had been nearly completely obliterated byfibrosis and concentric neo-osteogenesis, with tiny remnant of a lumen(FIG. 4), while at three weeks complete obliteration is evident (FIG. 5,low power).

The remaining portions of the sinuses were fully covered by epithelium.Forty-one point six (41.6%) ( 20/48) of the remaining posterior leftsinus was composed of squamous epithelium, with small areas ofdifferentiation (cuboidal or cylindrical ciliated epithelia). On theright sinus, 39.5% ( 19/48) of the posterior sections were covered by asquamous epithelium; but the anterior sections were almost completelycovered by a squamous epithelium (77%).

The effects of the present invention on sinonasal mucosal healing werethen investigated in this animal model. A slight modification of theprotocol used for the above studies included the insertion of anirrigating catheter into the nasal passages of the rabbits. Completemucosa stripping was performed with once daily irrigation of 3 cc(equivalent to 3 mL or about 0.1 oz.) of either saline solution or metalion solution for two weeks (FIG. 6).

Treatment with the Present Invention:

Pixel analysis of the complete mucosal stripping of sinuses (FIGS. 6B, Cand D) demonstrates that daily irrigation of the nasal passages with thepresent invention significantly inhibited osteoneogenesis compared toeither no treatment or treatment with normal saline (TABLE 10).

TABLE 10 pixels Rabbit Lumen Sinus Area % No Treatment 94,366 47396421.99 Daily irrigation with Normal Saline 336,846 4103053 8.21 Dailyirrigation with metal ion solution 1,083,097 3974206 27.25

Compared to daily saline irrigation, which demonstrated some inhibitionof osteoneogenesis compared to no treatment (8.21% vs 1.99%), dailyirrigation with present invention Solution 2 (sterile isotonicformulation of K+, Rb+, Ca2+, Zn2+ without preservative) resulted insignificant increase of the lumen (27.25%).

Example 2 Enhancement of Post-Surgical Sinonasal Recilialization

In addition to decreasing osteoneogenesis, daily irrigation with metalion solutions also increase the rate of recilialization of the medialwall in sinonasal passages in the rabbit compared to daily salineirrigation.

In a follow-up experiment, nine New Zealand White rabbits underwentbilateral medial wall maxillary mucosal stripping followed by placementof maxillary sinus indwelling irrigating catheters. In this way, eachnostril in each rabbit could be irrigated with different solutions for14 days.

Both the sides of the nose, and the therapy were randomized in therabbits as follows: one side of the nose in each rabbit was irrigatedonce daily (3 cc) with normal saline solution to act as a control forthe other side. In five of the rabbits the other side of the nose wasirrigated once daily (3 cc) with metal ion Solution 2 (sterile isotonicformulation of K⁺, Rb⁺, Ca²⁺, Zn²⁺ without preservative) and in theother four rabbits, the other side of the nose was irrigated daily (3cc) with metal ion solution 3 (sterile isotonic formulation of K⁺, Rb⁺,Ca²⁺, Zn²⁺, Mg²⁺, Br⁻ without preservative).

At the completion of the therapeutic trial, snouts were harvested andremucosalization with ciliated cells was assessed at the site of injuryby hematoxylin and eosin (H&E) staining as well as immunohistochemistryfor type IV tubulin, a marker of motile cilia. Semi quantitative gradingof reciliation was done by two blinded observers [0=no cilia; 1=<30%cilia; 2=30-60% cilia; 3=>60% cilia]. Statistical significance inciliation was determined using a one-tailed unpaired Student's t-test.

FIG. 7 shows a representative rabbit snout outlined byimmunohistochemistry using type IV-tubulin staining. The positions onthe medial walls of each snout where four evenly distributed images wereobtained for both H&E and type IV-tubulin (cilia) staining areindicated. FIG. 8 is an image taken from the uninjured lateral wall anddepicts normal mucosal ciliation. FIGS. 9 and 10 are images that arestained with type-IV b-tubulin from the upper and lower medial wall 14days after surgery and treatment with either saline (FIGS. 9A and 10A)or an isotonic formulation of K⁺, Rb⁺, Ca²⁺, and Zn²⁺ without thepreservative potassium sorbate (Solution 2) (FIGS. 9B and 10B).

The results of remucosalization with ciliated cells was assessed at thesite of injury by two blinded observers of hematoxylin and eosin (H&E)stained sections as well as immunohistochemistry for type IV tubulin, amarker of motile cilia. The sections were graded 0, 1, 2, or 3 accordingto the density of cilia in the sections and the results are summarizedin TABLE 11.

TABLE 11 Treatment Saline Solution 2^(a) Solution 3^(b) Type of Staining(n = 9) (n = 5) (n = 4) H & E^(c) 1.33 2.43 2.72 Type IV β-Tubulin 1.682.33 2.88 ^(a)Sterile isotonic formulation of K⁺, Rb⁺, Ca²⁺, Zn²⁺without preservative ^(b)Sterile isotonic formulation of K⁺, Rb⁺ Ca²⁺,Zn²⁺, Mg²⁺, Br⁻ without preservative ^(c)Hematoxylin and eosin

Example 3

Based upon the results of Example 2, another study using 24 rabbits wasperformed in which Solution 3 was compared to Normal Saline, the normalstandard of care for nasal irrigation treatment after FunctionalEndoscopic Sinus Surgery (FESS) in patients with chronic rhinosinusitis.Twenty four New Zealand White rabbits underwent bilateral medial wallmaxillary mucosal stripping followed by placement of an indwellingirrigation catheter as described above. In a randomized fashion, onenasal passage received 3 cc of normal saline daily, while thecontralateral nasal passage received 3 cc of Solution 3 daily. After a14 day therapeutic trial, snouts were harvested and remucosalization wasassessed by hematoxylin and eosin staining and immunohistochemicalstaining using type IV-tubulin, a marker of motile cilia. Reciliationgrading was as described above using two blinded observers. FIG. 11shows both H&E and immunohistochemical staining of sections of themedial maxillary sinus wall that had been surgically stripped of mucosalcells and treated for 14 days with either normal standard of care(saline), or solution 3. As can be seen, treatment using Solution 3results in a significantly accelerated and enhanced healing of themedial wall. The frequency distribution of scores for remucosalizationof the denuded medial maxillary sinuses with ciliated cells is given inTABLE 12.

TABLE 12 Type of Cilial Density Score Type of Staining Treatment 0% <30%30-60% >60% Hematoxylin & Eosin Saline 53 46 31 62 Solution 3 3 2 12 175Type IV β-Tubulin Saline 53 46 18 75 Solution 3 3 1 2 186

Hematoxylin and Eosin staining demonstrated that normal saline (n=24),the present standard of care for post-surgical treatment of chronicrhinosinusitis, had substantial bare areas with predominant ciliationscores under 30%. The group treated with Solution 3 (n=24) achieved astatistically significant improvement (using a Chi-square test tocompare the frequency distributions in each group) in ciliary density(>60%) when compared with normal saline (p<0.01). These results wereconfirmed with Type IV β-Tubulin staining Success of FunctionalEndoscopic Sinus Surgery (FESS) depends on restoration of normalmucociliary clearance. Daily topical irrigation with Solution 3 (sterileisotonic formulation of K⁺, Rb⁺, Ca²⁺, Zn²⁺, Mg²⁺, Br⁻) significantlyenhances ciliated remucosalization compared to saline.

Example 5

An animal model to characterize the re-epithelialization of the rabbitmaxillary sinus mucosa after surgical trauma was developed and thesafety and efficacy of the wound repair accelerative topicalpreparation, with Solutions 2 or 3, was tested (Entent Care, Maitland,Fla.).

Materials and Methods Solutions

A description of the original solution (Solution 1) can be found in U.S.Pat. No. 6,149,947, herein incorporated in its entirety by reference.Solution 1 is a specific hypertonic sinonasal formulation from aproprietary blend of mineral salts including potassium, calcium, zinc,and rubidium.

The inventors have developed several new solutions to enhance woundhealing, particularly in the sinonasal mucosa. Solution 2 is a sterileisotonic formulation containing K⁺, Rb⁺, Ca²⁺, and Zn²⁺ without apreservative. Solution 3 is a sterile isotonic formulation containingK⁺, Rb⁺, Ca²⁺, Zn²⁺, Mg²⁺, SO₄ ²⁻, and Br⁻ without a preservative.

Evaluation of Respiratory Ciliotoxicity

Mouse nasal septal explants were harvested as previously described.(Antunes M B, Woodworth B A, Bhargave G, et al. Murine nasal septa forrespiratory epithelial air-liquid interface cultures. Biotechniques.2007; 43:195-204). Once harvested, the septal mucosal explants wereplaced in a glass perfusion chamber on a thermostatically controlledstage, held in place with a nylon grid (1.5 mm) whose outer frame wassnapped into the inside of the perfusion chamber as previouslydescribed. (Schipor I, Palmer J N, Cohen A S, et al. Quantification ofciliary beat frequency in sinonasal epithelial cells using differentialinterference contrast microscopy and high-speed digital video imaging.Am J Rhinol 2006; 20:124-127).

The chamber was filled with Locke Ringer's solution (136 mM NaCl, 5.6 mMKCl, 10 mM HEPES, 14.3 mM NaHCO3, 1.2 mM MgCl2, 2.2 mM CaCl2, and 11.5mM dextrose, pH 7.35) and the temperature maintained between 35.5° C.and 37° C. with a dual channel heater (Warner Inst, Hamden, Conn.).Images were visualized using a Leica DMLFSA microscope set on an airtable (TMC, Peabody, Mass.) using a water immersion 363 objective anddifferential interference contrast (DIC) optics (Leica Microsystems,Bannockburn, Ill.).

Images were captured using a model A602f-2 Basler high speedmonochromatic digital video camera (Basler A G, Ahrensburg, Germany) ata sampling rate of 100 frames per second with a resolution of 640 3 480pixels. The video images were analyzed using the Sisson-Ammons VideoAnalysis (SAVA) system version 2.1.18. For each experiment, beatingcilia on the edge of the explant were detected with an uprightmicroscope. The digital image signal was then routed from the cameradirectly into a digital image acquisition board (National Instruments)within a Dell XPS 710 Workstation running Windows XP Professionaloperating system. Images were captured, compressed, and stored to disk.Files were then reloaded and analyzed with virtual instrumentationsoftware highly customized to perform CBF analysis. Recordings were madeevery 30 seconds for a total of 20 minutes.

Beating cilia were observed and recorded every 30 seconds for CBFdetermination. Once a stable baseline had been obtained (5 minutes), thetest solution, either Solution 2 or Solution 3 (with or without 0.075%potassium sorbate as a preservative), was substituted for the LockeRinger's solution while maintaining visualization of the beating cilia.Recordings continued for 10 minutes followed by replacement of the testsolution with Locke Ringer's for an additional 5 minutes. Each solutionwas tested in duplicate.

In Vivo Efficacy of Solution 2 to Accelerate Mucosal Healing

Prior to any animal experimentation, approval was obtained from theInstitutional Animal Care and Use Committee at the Philadelphia VeteransAffairs Medical Center (PVAMC). Nine New Zealand white rabbits wereused. Exposure of the maxillary sinus mucosa bilaterally was performedas previously described. (Chiu A G, Antunes M B, Palmer J N, et al.Evaluation of the in vivo efficacy of topical tobramycin againstPseudomonas sinonasal biofilms. J Antimicrob Chemother. 2007;59:1130-1134; Bleier B S, Palmer J N, Gratton M A, et al. In vivo lasertissue welding in the rabbit maxillary sinus. Am J Rhinol 2008; 22:625-628; Bleier B S, Palmer J N, Sparano A M, et al. Laser-assistedcerebrospinal fluid leak repair: an animal model to test feasibility.Otolaryngol Head Neck Surg. 2007; 137:810-814).

Briefly, following a midline nasal dorsum skin incision, medially basedperiosteal flaps were elevated bilaterally. The anterior face of themaxillary sinus was removed with an otologic drill (XPS-3000, MedtronicXomed Inc, Jacksonville, Fla.). Under microscopic visualization, a131-cm mucosal flap was elevated, using otologic instruments, off themedial wall of the maxillary sinus and removed. Bilateral maxillarysinus indwelling irrigating catheters were then placed and secured aspreviously described. (Chiu A G, Antunes M B, Palmer J N, et al.Evaluation of the in vivo efficacy of topical tobramycin againstPseudomonas sinonasal biofilms. J Antimicrob Chemother. 2007;59:1130-1134; Tamashiro E, Banks C A, Chen B, et al. In vivo effects ofcitric acid/zwitterionic surfactant cleansing solution on rabbit sinusmucosa. Am J Rhinol Allergy. 2009; 23:597-601).

Approximately 1.0 cm of tubing was placed into the sinus; the remainderwas tunneled under the skin and brought out through a stab incision atthe vertex of the cranium between the ears of the rabbit. Along thecourse of the catheter, vicryl sutures were used to drape the periosteumover the catheter. The hub of the tubing was capped and secured to theskin with a purse string suture of 3-0 nylon, and the midline incisionwas closed with a running 3-0 nylon. Patency of the catheter was checkedby instilling 3 mL of normal saline (NS) or test solution and confirmingdrainage through the ipsilateral naris. Randomization of sinuses wasperformed prior to instillation of any solution. For each rabbit, oneside received NS while the other side received either Solution 2 orSolution 3. Thus, each rabbit served as its own “saline” control,whereas the sides were randomized. Each sinus was irrigated daily with 3mL of solution for 14 days. At the completion of the therapeutic trial,the rabbits were euthanized using sedation and an intracardiacbarbiturate overdose.

Snouts were harvested, fixed for 24 hours in 10% normal bufferedformalin, and then decalcified for 48 hours in rapid decalcifier(Electron Microscopy Sciences, Hatfield, Pa.). After sectioning,dehydrating, and paraffin embedding, the tissue was sectioned, andremucosalization with ciliated cells was assessed at the site of injuryby hematoxylin and eosin (H&E) staining as well as immunohistochemistry(IHC) for type-IV β-tubulin, a marker of motile cilia.

The stripping was performed in the middle third of the medial wall ofthe maxillary sinuses, and 4 representative segments of regeneratedmucosa were imaged at 340 magnification from each side. The micrographsfrom both H&E and IHC staining were assembled in random order andreviewed by 2 blinded trained observers. Thus, for each sinus evaluated,8 data points for H&E and 8 data points for type IV β-tubulinimmunohistochemistry were generated.

A semi quantitative grading of reciliation was used based on the amountof mucosa covered with cilia: 0=no cilia, 1=less than 30% cilia, 2=30%to 60% cilia, 3=greater than 60% cilia (FIG. 12). Given the categoricalnature of the data sets of reciliation, the inventors applied thechi-square test to determine significance between each group using Prism5 (GraphPad, La Jolla, Calif.). The animal protocol was IACUC approved.

Results Ciliotoxicity Analysis

Ciliary beat frequency of murine septal mucosal explants was observedand quantified for 5 minutes in Locke Ringer's solution. The bathingsolution was exchanged with Solution 2 (up arrow FIG. 13A, blacktracings), and ciliary beat frequency was continuously monitored for anadditional 10 minutes. Following these treatments, the bathing solutionwas once again exchanged to Locke Ringer's (down arrow FIG. 13, blacktracings). The experiment was repeated with the addition of thepreservative potassium sorbate (0.075%) (FIG. 13, gray tracings).Because each explant has a slightly different intrinsic basal ciliarybeat frequency, the tracings were normalized by averaging thepretreatment baseline frequencies (T=0 to T=5 minutes) and dividing eachindividual frequency by the average basal frequency (fPHI/fbasal) (FIG.13 b).

As demonstrated in FIG. 13, the addition of Solution 2 had no effect onbasal ciliary beat frequency. It should be noted that immediately afterexchanging solutions (up and down arrow), there was an increase inciliary beat frequency, which was most likely caused by mechanicalperturbation of the epithelium and subsequent activation ofmechanosensors. (Winters S L, Davis C W, Boucher R C. Mechanosensitivityof mouse tracheal ciliary beat frequency: roles for Ca21, purinergicsignaling, tonicity, and viscosity. Am J Physiol Lung Cell Mol Physiol.2007; 292:L614-L624). Similar analysis was performed using Solution 3.As demonstrated in FIG. 14A, the addition of these elements had nodetrimental effects on murine septal ciliary beat frequency.

Sinonasal Mucosal Regeneration

Mucosal stripping of the medial wall of the rabbit maxillary sinus withsubsequent daily topical treatments for 14 days was performed asdescribed above. A total of 18 maxillary sinuses were analyzed.Representative photomicrographs of H&E-stained sections ofsaline-treated sinuses versus sinuses treated with Solution 2 orSolution 3 clearly demonstrate a profound effect of the solutions onmucosal regeneration (FIG. 15A). A similar effect of Solutions 2 and 3on type IV β-tubulin immunohistochemistry is demonstrated in FIG. 15B.

Compiling all the data by frequency of distribution, Table 13demonstrates significantly more scores of 3 in the sinuses treated withSolutions 2 and 3 as compared with those treated with saline as assessedby H&E staining as well as type IV β-tubulin immunohistochemistry(P<0.01). The median score for each treatment arm and analysis confirmsthis finding (Table 14). Furthermore, the percentage of the scoringbreakdown for both outcome analyses illustrates the shift to nearcomplete ciliation (score of 3) in the sinuses treated with Solutions 2and 3 compared with the saline treated sinuses (FIG. 16).

TABLE 13 Frequency of Distribution and Percentage of Reciliation ScoreReciliation Score 0 1 2 3 Total H&E Saline 20 (28) 24 (33) 12 (17) 16(22) 72 (100) Solution 2  4 (10) 0 (0) 11 (28) 25 (62) 40 (100) Solution3 0 (0) 2 (6)  5 (16) 25 (78) 32 (100) Type IV β-tubulin staining Saline14 (19) 18 (25) 17 (24) 23 (32) 72 (100) Solution 2  5 (12) 1 (3) 10(25) 24 (60) 40 (100) Solution 3 0 (0) 0 (0)  4 (12) 28 (88) 32 (100)H&E, hematoxylin and eosin. Values are frequency of distribution(percentage for each score as a function of the total observations). Atotal of 72, 40, and 32 photomicrographs were analyzed for saline,Solution 2 and Solution 3, respectively for each evaluation of ciliationwith distribution by grade for each treatment arm.

TABLE 14 Median Reciliation Score Saline Solution 2 Solution 3 H&E 1 3 3β-tubulin IHC 2 3 3 H&E hematoxylin and eosin; Enhanced reciliationfollowing application of Solutions 2 and 3 is evident by the medianscore.

The mechanism of action of the cation-containing solutions presentedherein appear to be aimed at modulating MMP activity, since the use of acation-containing solution has previously been found to decreaseexpression of MMP messenger RNA while increasing the expression of TIMPRNA in cultured fibroblasts. (Pirayesh A, Dessy L A, Rogge F J, et al.The efficacy of a polyhydrated ionogen impregnated dressing in thetreatment of recalcitrant diabetic foot ulcers: a multi-centre pilotstudy. Acta Chir Belg. 2007; 107:675-681). The inventors investigatedwhether Solutions 2 and 3 have any applicability in modulating thehealing process of the respiratory mucosa following functionalendoscopic sinus surgery.

Evaluation of the Solutions 2 and 3 with or without potassium sorbate asa preservative demonstrated no ciliotoxicity in the explant model. Thisis an important initial screening step in any potential sinonasaltopical therapy, as very promising in vitro solutions have demonstratedsignificant ciliotoxicity. (Tamashiro E, Banks C A, Chen B, et al. Invivo effects of citric acid/zwitterionic surfactant cleansing solutionon rabbit sinus mucosa. Am J Rhinol Allergy. 2009; 23:597-601).Furthermore, because preservatives added to nasal preparations have alsodemonstrated varying degrees of ciliotoxicity, the inventors screenedthe combinations of the solutions with or without the preservativepotassium sorbate for potential synergistic ciliotoxicty. (Batts A H,Marriott C, Martin G P, et al. The effect of some preservatives used innasal preparations on mucociliary clearance. J Pharm Pharmacol. 1989;41:156-159).

To evaluate for mucosal regeneration, the inventors modified thepreviously reported rabbit maxillary sinus irrigation model to include areproducible mucosal injury of the medial wall of the maxillary sinus.(Chiu A G, Antunes M B, Feldman M, et al. An animal model for the studyof topical medications in sinusitis. Am J Rhinol 2007; 21:5-9). The datademonstrate that 2 weeks of treatment with daily irrigation of eitherSolution 2 or Solution 3 compared with saline statistically significantenhanced reciliation of the injury site. Thus, Solutions 2 and 3 holdtremendous promise as a postsurgical irrigation, especially in caseswhere significant bone exposure (purposeful or inadvertent) is evidentat the conclusion of the surgical procedure.

As shown in Table 15, below, treatment with either Solutions 2 or 3 hada significant effect on reciliation and healing in the sinonasal mucosaas compared to treatment with saline alone.

TABLE 15 H&E Type IV β-Tubulin Saline 1.33 1.68 (n = 9) Solution 2 2.432.33 (n = 5) Solution 3 2.72 2.88 (n = 4)

Example 4

The above prior safety studies demonstrated that the solution did notinduce any ciliotoxicity. Additionally, as part of the safety study, asmall pilot study was completed comparing Solutions 2 and 3 to saline onsinonasal mucosal healing. Following a 2-week trial with dailyirrigation, the sinuses treated with Solutions 2 or 3 demonstratedsignificant improvement in mucosal regeneration compared to saline.Based on this pilot study, a new prospective randomized blinded animalstudy was designed to detect an effect size of 40%, with an 80% powerand significance level of 5%.

Subjects and Methods Power Calculation

Based on data from a previous study that demonstrated a significantimprovement in paranasal mucosal reciliation following application oftopical Solution 2 or Solution 3, a new prospective randomized blindedanimal study was designed to detect an effect size of 40%, with an 80%power and significance level of 5%, using Epi Info, a Centers forDisease Control (CDC) statistical tool, a sample size of 24 rabbits wascalculated. (DePoortere D, Kofonow J M, Chiu A G, et al. Enhanced postsurgical remucosalization in a rabbit model. Otolaryngol Head Neck Surg.2010; 143:P129).

Solutions

Solution 3 is a sterile isotonic formulation containing K⁺, Rb⁺, Ca²⁺,Zn²⁺, Mg²⁺, SO₄ ²⁻, and Br⁻ without a preservative.

In Vivo Efficacy of Solution 3 to Accelerate Mucosal Healing

Prior to any animal experimentation approval was obtained from theInstitutional Animal Care and Use Committee at the Philadelphia VeteransAffairs Medical Center (PVAMC). Twenty-four New Zealand white rabbitswere used. Exposure of the maxillary sinus mucosa bilaterally wasperformed as previously described. (Chiu A G, Antunes M B, Palmer J N,et al. Evaluation of the in vivo efficacy of topical tobramycin againstPseudomonas sinonasal biofilms. J Antimicrob Chemother. 2007;59:1130-1134; Bleier B S, Palmer J N, Gratton M A, et al. In vivo lasertissue welding in the rabbit maxillary sinus. Am J Rhinol. 2008;22:625-628; Bleier B S, Palmer J N, Sparano A M, et al. Laser assistedcerebrospinal fluid leak repair: an animal model to test feasibility.Otolaryngol Head Neck Surg. 2007; 137:810-814).

Briefly, following a midline nasal dorsum skin incision, medially-basedperiosteal flaps were elevated bilaterally. The anterior face of themaxillary sinus was removed with an otologic drill (XPS-3000; Medtronic,Jacksonville, Fla.). Under microscopic visualization a 1-cm×1-cm mucosalflap was elevated off the medial wall of the maxillary sinus usingotologic instruments and removed. Bilateral maxillary sinus indwellingirrigating catheters were then placed and secured as previouslydescribed. (Chiu A G, Antunes M B, Palmer J N, et al. Evaluation of thein vivo efficacy of topical tobramycin against Pseudomonas sinonasalbiofilms. J Antimicrob Chemother. 2007; 59:1130-1134; Tamashiro E, BanksC A, Chen B, Gudis D A, Doghramji L, Myntti M, Chiu A G, Cohen N A. Invivo effects of citric acid/zwitterionic surfactant (CAZS) cleansingsolution on rabbit sinus mucosa. Am J Rhinol Allergy. 2009; 23:597-601).Approximately 1.0 cm of tubing was placed into the sinus and theremainder was tunneled under the skin of the forehead, and brought outthrough a stab incision at the vertex of the cranium between the ears.Along the course of the catheter, vicryl sutures were used to drape theperiosteum over the catheter. The hub of the tubing was capped andsecured to the skin with a purse string suture of 3-0 nylon and themidline incision was closed with a running 3-0 nylon. Patency of thecatheter was checked by instilling 3 cc of normal saline (NS) orSolution 3 and confirming drainage through the ipsilateral nares.Randomization of sinus was performed prior to instillation of anysolution. For each rabbit, 1 side received NS while the other sidereceived Solution 3. Thus, each rabbit served as its own “saline”control, while the sides were randomized. Each sinus was irrigated dailywith 3 cc of solution for 14 days. At the completion of the therapeutictrial, rabbits were euthanized using sedation and an intracardiacbarbiturate overdose.

Snouts were harvested, fixed for 24 hours in 10% normal bufferedformalin, and then decalcified for 48 hours in rapid decalcifier(Electron Microscopy Sciences, Hatfield, Pa.). After sectioning,dehydrating, and paraffin embedding, the tissue was sectioned andremucosalization with ciliated cells was assessed at the site of injuryby hematoxylin and eosin (H&E) staining as well as immunohistochemistry(IHC) for type IV β-tubulin, a marker of motile cilia.

The stripping was performed in the middle third of the medial wall ofthe maxillary sinuses, 4 representative segments of regenerated mucosawere imaged at ×40 magnification from each side. The micrographs fromboth H&E and IHC staining were assembled in random order and reviewed by2 blinded trained observers. A semi quantitative grading of reciliationwas used based on the amount of mucosa covered with cilia (0=no cilia;1=<30% cilia; 2=31-60% cilia; 3=>60% cilia) as previously described.(DePoortere D, Kofonow J M, Chiu A G, et al. Enhanced post surgicalremucosalization in a rabbit model. Otolaryngol Head Neck Surg. 2010;143:P129).

A chi-square test was used to compare the distributions in each groupand determine significance.

Results

To illustrate the experimental paradigm, a montage of a rabbit snout wasassembled demonstrating the surgical antrostomy locations, region alongthe medial wall of mucosal stripping, as well as the approximate regionsof the 4 random photomicrographs obtained for evaluation (FIG. 17).

The degree of mucosal regeneration was scored as described above withrepresentative photomicrographs of each grade by H&E staining and typeIV β-tubulin IHC displayed in FIG. 18.

Mucosal stripping of the medial wall of the rabbit maxillary sinus withsubsequent daily topical treatments for 14 days was performed asdescribed above. A total of 48 maxillary sinuses were analyzed with 4photomicrographs of each sinus obtained yielding a total of 192 images.Representative photomicrographs of H&E stained sections (FIG. 19A) aswell as type IV tubulin immunohistochemistry (FIG. 19B) of salinetreated vs. Solution 3-treated sinuses clearly demonstrate a profoundeffect of Solution 3 on mucosal regeneration. The Solution 3-treatedsinuses demonstrate normal appearing respiratory mucosa with an intactbrush border compared to the saline-treated sinuses demonstrating sparseciliation and flattened fibroblastic appearing cells.

A frequency of distribution table for all 192 images demonstrates aprofound shift toward complete reciliation in Solution 3 (Table 16). H&Estaining comparison demonstrated NS-treated sinuses (n=24) hadsubstantial bare areas with predominant ciliation scores under 33%, only93 images of 192 (48.43%) were scored above 66%. The Solution 3-treatedgroup (n=24) achieved a statistically significant improve in ciliarydensity, 175 pictures out of 192 (91.14%) were scored above 66%,chi-square value of 147.25 (p<0.01) (FIG. 19A). These results wereconfirmed with a cilia specific IHC staining for Type IV β-tubulin,where the frequency distribution again evinced significant lowerciliation in the NS-treated group when compared with the Solution3-treated sinuses, chi-square 147.73 (p<0.01) (FIG. 19B). The medianscore for the Solution 3-treated sinuses was 3 for both H&E and type IVβ-tubulin IHC compared to 1 for both analyses of the saline-treated(p<0.01).

Additionally, the initial data illustrated the efficacy of Solution 2 inaccelerating the sinonasal mucosal healing process. An effect size of40%, with an 80% power and significance level of 5% was used fordetection. Utilizing 24 rabbits, and based on our semi-quantitativescoring system for ciliated mucosal regeneration, the inventorsdemonstrate a robust difference in sinonasal wound healing, includingdebridement of damaged tissue, following a 2-week course of daily sinusirrigation with Solution 3 as compared to NS. It is important to notethat this model does not take into account infection, or pre-existinginflammation as is often encountered in CRS patients undergoing surgicalintervention.

TABLE 16 Frequency and Distribution of Scores for H&E andImmunohistochemistry for Both Treatments for all Rabbits 0% <30%30%-60% >60% H&E Saline 53 46 31 62 Solution 3 3 2 12 175 Tubulin Saline53 46 18 75 Solution 3 3 1 2 186 *A total of 192 photomicrographs wereanalyzed for each evaluation of ciliation with distribution by grade foreach treatment arm. H&E = hematoxylin and eosin;

Example 6 Materials and Methods Solutions

For these experiments, Solutions 3 and 4 were compared to saline in arabbit model for sinonasal mucosal injury. Solution 3 is a sterileisotonic formulation containing K⁺, Kb⁺, Ca²⁺, Zn²⁺, Mg²⁺, SO₄ ²⁻, andBr⁻ without a preservative. Solution 4 is a sterile isotonic formulationcontaining Mg²⁺, Br⁻, Na+, Cl−, and SO₄ ²⁻ without a preservative.

Prior to any animal experimentation approval was obtained from theInstitutional Animal Care and Use Committee at the Philadelphia VeteransAffairs Medical Center (PVAMC). Six New Zealand white rabbits were used.Exposure of the maxillary sinus mucosa bilaterally was performed aspreviously described. (Chiu A G, Antunes M B, Palmer J N, et al.Evaluation of the in vivo efficacy of topical tobramycin againstPseudomonas sinonasal biofilms. J Antimicrob Chemother. 2007;59:1130-1134; Bleier B S, Palmer J N, Gratton M A, et al. In vivo lasertissue welding in the rabbit maxillary sinus. Am J Rhinol. 2008;22:625-628; Bleier B S, Palmer J N, Sparano A M, et al. Laser assistedcerebrospinal fluid leak repair: an animal model to test feasibility.Otolaryngol Head Neck Surg. 2007; 137:810-814).

Briefly, following a midline nasal dorsum skin incision, medially-basedperiosteal flaps were elevated bilaterally. The anterior face of themaxillary sinus was removed with an otologic drill (XPS-3000; Medtronic,Jacksonville, Fla.). Under microscopic visualization a 1-cm×1-cm mucosalflap was elevated off the medial wall of the maxillary sinus usingotologic instruments and removed. Bilateral maxillary sinus indwellingirrigating catheters were then placed and secured as previouslydescribed. (Chiu A G, Antunes M B, Palmer J N, et al. Evaluation of thein vivo efficacy of topical tobramycin against Pseudomonas sinonasalbiofilms. J Antimicrob Chemother. 2007; 59:1130-1134; Tamashiro E, BanksC A, Chen B, Gudis D A, Doghramji L, Myntti M, Chiu A G, Cohen N A. Invivo effects of citric acid/zwitterionic surfactant (CAZS) cleansingsolution on rabbit sinus mucosa. Am J Rhinol Allergy. 2009; 23:597-601).Approximately 1.0 cm of tubing was placed into the sinus and theremainder was tunneled under the skin of the forehead, and brought outthrough a stab incision at the vertex of the cranium between the ears.Along the course of the catheter, vicryl sutures were used to drape theperiosteum over the catheter. The hub of the tubing was capped andsecured to the skin with a purse string suture of 3-0 nylon and themidline incision was closed with a running 3-0 nylon. Patency of thecatheter was checked by instilling 3 cc of normal saline (NS) orSolution 3 or 4 and confirming drainage through the ipsilateral nares.Randomization of sinus was performed prior to instillation of anysolution. For each rabbit, 1 side received NS while the other sidereceived either Solution 3 or Solution 4. Thus, each rabbit served asits own “saline” control, while the sides were randomized. Each sinuswas irrigated daily with 3 cc of solution for 14 days. At the completionof the therapeutic trial, rabbits were euthanized using sedation and anintracardiac barbiturate overdose.

One of the six rabbits had no first surgery due to neurological issuesbut was used as a time 0 for stripping. The remaining 5 rabbits,illustrating 10 sinuses, were used to demonstrate the efficacy ofSolution 3 versus Solution 4 with saline used as a control. The salinecontrol was administered to 2 sinuses; Solution 3 was administered to 3sinuses; and Solution 4 was administered to 5 sinuses.

Snouts were harvested, fixed for 24 hours in 10% normal bufferedformalin, and then decalcified for 48 hours in rapid decalcifier(Electron Microscopy Sciences, Hatfield, Pa.). After sectioning,dehydrating, and paraffin embedding, the tissue was sectioned andremucosalization with ciliated cells was assessed at the site of injuryby hematoxylin and eosin (H&E) staining.

The stripping was performed in the middle third of the medial wall ofthe maxillary sinuses, 4 representative segments of regenerated mucosawere imaged at ×40 magnification from each side. The micrographs fromH&E staining were assembled in random order and reviewed by 2 blindedtrained observers. A semi quantitative grading of reciliation was usedbased on the amount of mucosa covered with cilia (0=no cilia; 1=<30%cilia; 2=31-60% cilia; 3=>60% cilia) as previously described.(DePoortere D, Kofonow J M, Chiu A G, et al. Enhanced post surgicalremucosalization in a rabbit model. Otolaryngol Head Neck Surg. 2010;143:P129).

A chi-square test was used to compare the distributions in each groupand determine significance.

Results

Representative photomicrographs of H&E stained sections of salinetreated (FIG. 20) vs. Solution 3-treated sinuses (FIG. 21) vs. Solution4-treated sinuses (FIG. 22) clearly demonstrate a profound effect ofSolutions 3 and 4 on mucosal regeneration. The Solution 3-treated andSolution 4-treated sinuses demonstrate normal appearing respiratorymucosa with an intact brush border compared to the saline-treatedsinuses demonstrating sparse ciliation and flattened fibroblasticappearing cells.

TABLE 17 H&E Saline 1.42 (n = 2) Solution 4 2.73 (n = 5) Solution 3 2.94(n = 3)

In comparing the results in Table 17 to the results in Table 15, thereseems to be at least an additive effect, if not a synergistic effect, ofthe addition of the Mg²⁺, Br⁻ and possibly the Na⁺, Cl⁻, and SO₄ ²⁻ ionsin Solution 3 (containing K⁺, Rb⁺, Ca²⁺, Zn²⁺, Na⁺, Cl⁺, and SO₄ ²⁻,Mg²⁺, and Br⁻) as compared to Solution 2 containing only K⁺, Rb⁺, Ca²⁺,and Zn²⁺.

Unexpectedly, Solution 4 containing only Mg²⁺, Br⁻, Na⁺, Cl⁻, and SO₄ ⁻ions seemed to perform at least as well as if not even better thanSolution 2. These results seem to indicate that Solutions 3 and 4 havepotential to be used for promoting sinus wound healing, includingdebridement of damaged tissue, after sinus surgery as illustrated inFIGS. 21 and 22. In addition, both solutions demonstrated enhancedciliated remucosalization as also illustrated in FIGS. 21 and 22.

CONCLUSION

Advances in surgical instrumentation have improved the rhinologist'sability to practice mucosal preservation surgery. It is well acceptedthat mucosal stripping leads to poor healing of the respiratory mucosawith loss of ciliary function in critical areas resulting in adverseclinical outcomes. However, inadvertent mucosal stripping does occur aswell as clinical situations dictating intentional mucosal resection.Solutions 3 and 4 were shown to be capable of debriding damaged tissueto enhance wound healing as well as enhance mucosal reciliation in thesinonasal mucosa. In the rabbit model, daily irrigation with Solutions2, 3, or 4 enhanced ciliated remucosalization compared to saline, thusillustrating these solutions have applicability in promoting sinus woundhealing after sinus surgery.

In the preceding specification, all documents, acts, or informationdisclosed does not constitute an admission that the document, act, orinformation of any combination thereof was publicly available, known tothe public, part of the general knowledge in the art, or was known to berelevant to solve any problem at the time of priority.

The disclosures of all publications cited above are expresslyincorporated herein by reference, each in its entirety, to the sameextent as if each were incorporated by reference individually.

It will be seen that the advantages set forth above, and those madeapparent from the foregoing description, are efficiently attained andsince certain changes may be made in the above construction withoutdeparting from the scope of the invention, it is intended that allmatters contained in the foregoing description or shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

It is also to be understood that the following claims are intended tocover all of the generic and specific features of the invention hereindescribed, and all statements of the scope of the invention which, as amatter of language, might be said to fall there between. Now that theinvention has been described,

What is claimed is:
 1. A method of promoting mucosal reciliation in asubject consisting essentially of: administering an effective amount ofa sterile isotonic solution to a subject in need thereof comprising: atherapeutically effective amount of potassium ions; a therapeuticallyeffective amount of rubidium ions; a therapeutically effective amount ofcalcium ions; a therapeutically effective amount of zinc ions; atherapeutically effective amount magnesium ions; a therapeuticallyeffective amount of bromide ions; a therapeutically effective amount ofsulfate ions; a therapeutically effective amount of citric acid; and apharmaceutically acceptable carrier; wherein the administration of theisotonic solution causes mucosal reciliation.
 2. The method of claim 1,wherein the isotonic solution is administered at least once daily. 3.The method of claim 1, wherein the isotonic solution is administered tothe subject for at least 14 days.
 4. The solution of claim 1, whereintonicity of the solution is between about 0.9% and about 3.6%.
 5. Thesolution of claim 1, wherein the pharmaceutically acceptable carrier iswater.
 6. The solution of claim 1, wherein the amount of potassium ionsis between about 0.02% to about 3.0% (w/v).
 7. The solution of claim 1,wherein the amount of rubidium ions is between about 0.0001% to about4.0% (w/v).
 8. The solution of claim 1, wherein the amount of calciumions is between about 0.0000055 to about 1.3% (w/v).
 9. The solution ofclaim 1, wherein the amount of zinc ions is between about 0.0000057% toabout 2.12% (w/v).
 10. The solution of claim 1, wherein the amount ofmagnesium ions is between about 0.0075 to about 1.14% (w/v).
 11. Thesolution of claim 1, wherein the amount of bromide ions is between about0.0002% to about 2.6% (w/v).